Dual targeting of brain region‐specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1
2021; Springer Nature; Volume: 40; Issue: 7 Linguagem: Inglês
10.15252/embj.2020106106
ISSN1460-2075
AutoresWon‐Seok Lee, Laura A. Lavery, Maxime W.C. Rousseaux, Eric B Rutledge, Youjin Jang, Ying‐Wooi Wan, Sih‐Rong Wu, Won‐Ho Kim, Ismael Al‐Ramahi, Smruti Rath, Carolyn J. Adamski, Vitaliy V. Bondar, Ambika Tewari, Shirin Soleimani, Samantha Mota, Hari Krishna Yalamanchili, Harry T. Orr, Zhandong Liu, Juan Botas, Huda Y. Zoghbi,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle11 March 2021free access Source DataTransparent process Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 Won-Seok Lee Won-Seok Lee orcid.org/0000-0001-8633-4682 Integrative Molecular and Biomedical Science Program, Baylor College of Medicine, Houston, TX, USA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Laura Lavery Laura Lavery Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Maxime W C Rousseaux Maxime W C Rousseaux orcid.org/0000-0002-2737-6193 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Eric B Rutledge Eric B Rutledge orcid.org/0000-0003-4785-652X Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Youjin Jang Youjin Jang Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Ying-Wooi Wan Ying-Wooi Wan Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Sih-Rong Wu Sih-Rong Wu Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Wonho Kim Wonho Kim Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Ismael Al-Ramahi Ismael Al-Ramahi Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Smruti Rath Smruti Rath Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Carolyn J Adamski Carolyn J Adamski Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Vitaliy V Bondar Vitaliy V Bondar Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Ambika Tewari Ambika Tewari Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Shirin Soleimani Shirin Soleimani Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Samantha Mota Samantha Mota Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Hari K Yalamanchili Hari K Yalamanchili Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Harry T Orr Harry T Orr orcid.org/0000-0001-6118-741X Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Zhandong Liu Zhandong Liu Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Juan Botas Juan Botas Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Huda Y Zoghbi Corresponding Author Huda Y Zoghbi [email protected] orcid.org/0000-0002-0700-3349 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Won-Seok Lee Won-Seok Lee orcid.org/0000-0001-8633-4682 Integrative Molecular and Biomedical Science Program, Baylor College of Medicine, Houston, TX, USA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Laura Lavery Laura Lavery Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Maxime W C Rousseaux Maxime W C Rousseaux orcid.org/0000-0002-2737-6193 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Eric B Rutledge Eric B Rutledge orcid.org/0000-0003-4785-652X Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Youjin Jang Youjin Jang Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Ying-Wooi Wan Ying-Wooi Wan Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Sih-Rong Wu Sih-Rong Wu Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Wonho Kim Wonho Kim Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Ismael Al-Ramahi Ismael Al-Ramahi Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Smruti Rath Smruti Rath Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Carolyn J Adamski Carolyn J Adamski Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Vitaliy V Bondar Vitaliy V Bondar Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Ambika Tewari Ambika Tewari Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Shirin Soleimani Shirin Soleimani Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Samantha Mota Samantha Mota Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Hari K Yalamanchili Hari K Yalamanchili Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Harry T Orr Harry T Orr orcid.org/0000-0001-6118-741X Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Zhandong Liu Zhandong Liu Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Juan Botas Juan Botas Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Search for more papers by this author Huda Y Zoghbi Corresponding Author Huda Y Zoghbi [email protected] orcid.org/0000-0002-0700-3349 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA Howard Hughes Medical Institute, Houston, TX, USA Search for more papers by this author Author Information Won-Seok Lee1,2,3, Laura Lavery2,3, Maxime W C Rousseaux2,3,8, Eric B Rutledge3,4, Youjin Jang2,3, Ying-Wooi Wan2,3, Sih-Rong Wu3,5, Wonho Kim3,6, Ismael Al-Ramahi2,3, Smruti Rath2,3, Carolyn J Adamski2,3,6, Vitaliy V Bondar2,3, Ambika Tewari2,3, Shirin Soleimani2,3, Samantha Mota2,3, Hari K Yalamanchili3,4, Harry T Orr7, Zhandong Liu3,4, Juan Botas2,3 and Huda Y Zoghbi *,2,3,4,6 1Integrative Molecular and Biomedical Science Program, Baylor College of Medicine, Houston, TX, USA 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 3Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA 4Department of Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, USA 5Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA 6Howard Hughes Medical Institute, Houston, TX, USA 7Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA 8Present address: Department of Cellular and Molecular Medicine, University of Ottawa Brain and Mind Research Institute, University of Ottawa, Ottawa, ON, Canada *Corresponding author. Tel: +1 713 798 6558; E-mail: [email protected] The EMBO Journal (2021)40:e106106https://doi.org/10.15252/embj.2020106106 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A critical question in neurodegeneration is why the accumulation of disease-driving proteins causes selective neuronal loss despite their brain-wide expression. In Spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded Ataxin-1 (ATXN1) causes selective degeneration of cerebellar and brainstem neurons. Previous studies revealed that inhibiting Msk1 reduces phosphorylation of ATXN1 at S776 as well as its levels leading to improved cerebellar function. However, there are no regulators that modulate ATXN1 in the brainstem—the brain region whose pathology is most closely linked to premature death. To identify new regulators of ATXN1, we performed genetic screens and identified a transcription factor-kinase axis (ZBTB7B-RSK3) that regulates ATXN1 levels. Unlike MSK1, RSK3 is highly expressed in the human and mouse brainstems where it regulates Atxn1 by phosphorylating S776. Reducing Rsk3 rescues brainstem-associated pathologies and deficits, and lowering Rsk3 and Msk1 together improves cerebellar and brainstem function in an SCA1 mouse model. Our results demonstrate that selective vulnerability of brain regions in SCA1 is governed by region-specific regulators of ATXN1, and targeting multiple regulators could rescue multiple degenerating brain areas. SYNOPSIS The molecular mechanisms of selective neuronal vulnerability in neurodegenerative disorders are largely unknown. Here we report that selective vulnerability in Spinocerebellar Ataxia Type 1 (SCA1, caused by an expansion of polyglutamine-encoding CAG repeat in ATXN1) is partially driven by brain region-specific regulators of the ATXN1 protein. Genetic screen identifies ZBTB7B as a novel ATXN1 regulator. ZBTB7B regulates ATXN1 via activating RSK3 transcription. RSK3 stabilizes ATXN1 protein by phosphorylating ATXN1 on S776 (also a target of MSK1 in the cerebellum). RSK3 is predominantly expressed in the brainstem whereas MSK1 is expressed in the cerebellum. Reducing RSK3 and MSK1 rescues brainstem and cerebellar neurodegeneration, respectively. Introduction Neurodegenerative disorders are incurable debilitating diseases resulting in irreversible deterioration of neuronal integrity. They are globally prevalent with the two most common diseases, Alzheimer's (AD) and Parkinson's disease (PD) affecting as many as 0.5% of the world population (Tysnes & Storstein, 2017; Erkkinen et al, 2018). In general, most neurodegenerative diseases are regarded as proteinopathies because across different disorders, accumulation of certain toxic proteins is commonly found in both familial and sporadic cases. For example, accumulation of β-amyloid (Aβ), α-synuclein, and tau are found in familial and sporadic cases of AD, PD, and frontotemporal dementia (FTD), respectively. Decreasing levels of the toxic proteins is considered to be beneficial as many preclinical studies have shown that this strategy can rescue various disease phenotypes (Kumar et al, 2000; Kordasiewicz et al, 2012; Spencer et al, 2016; Friedrich et al, 2018; Silva et al, 2019). One of the characteristic, and enigmatic, proteinopathic patterns in neurodegenerative disorders is that specific brain regions and/or neuronal cell types are vulnerable to pathological protein accumulations, despite the brain-wide expression of these proteins (Saxena & Caroni, 2011; Roselli & Caroni, 2015; Fu et al, 2018). To gain insight into mechanisms that contribute to brain region-specific vulnerability, we turned to a well-characterized and genetically defined neurodegenerative disorder: Spinocerebellar Ataxia type 1 (SCA1). SCA1 is a fatal late-onset neurodegenerative disorder caused by an expansion of CAG repeats encoding polyglutamine (polyQ) tract in the ATXN1 gene (Orr et al, 1993). Although ATXN1 is broadly expressed in the brain, cerebellar and brainstem neurons degenerate, causing loss of motor coordination, and premature death due to swallowing and breathing difficulties, respectively (Zoghbi & Orr, 2009). Neuronal toxicity of mutant ATXN1 is caused by a gain-of-function mechanism based on the following evidence: (i) Knockout of Atxn1 in mice does not cause neurodegeneration, whereas increase of even wild-type protein does (Matilla et al, 1998; Fernandez-Funez et al, 2000; Gennarino et al, 2015); (ii) polyQ expansion stabilizes ATXN1 by retarding its proteasomal degradation (Cummings et al, 1999); (iii) reducing ATXN1 by only 20% can rescue some disease phenotypes (Jafar-Nejad et al, 2011). Previous efforts have identified regulators that, when targeted, can decrease ATXN1 protein levels. However, to date, these approaches have succeeded in identifying ATXN1 regulators that when targeted only rescue cerebellar dysfunction. For example, MSK1 is a serine/threonine kinase that phosphorylates ATXN1 at serine 776 (S776), promoting its stability through its interaction with 14-3-3ε (Chen et al, 2003; Park et al, 2013). Loss of either Msk1 or 14-3-3ε decreases Atxn1 levels in cerebellum and rescues cerebellar related deficits, without impacting the brainstem and its associated pathologies (Jafar-Nejad et al, 2011; Park et al, 2013). Given that brainstem dysfunction greatly impacts survival of SCA1 patients, current therapeutic targets have limited promise for full efficacy and thus identifying ATXN1 regulators that when targeted rescue brainstem dysfunction are of high value. Using a forward genetic screen in cells, we found that a transcription factor ZBTB7B, and to a lesser degree its paralog ZBTB7A, alters ATXN1 levels. ZBTB7A and ZBTB7B are members of the BTB-ZF family of transcription factors harboring the protein-interaction BTB domain and DNA-binding ZF domains whose neuronal functions remain largely unknown (Siggs & Beutler, 2012). Our transcriptomic studies revealed that ZBTB7B modulates ATXN1 indirectly by regulating the expression of RPS6KA2, which encodes RSK3, and this ZBTB7B-RSK3 axis regulates ATXN1 in vivo. Although RSK and MSK belong to two distinct groups of kinases, we found that both phosphorylate ATXN1 at S776. However, unlike Msk1, Rsk3 predominantly regulates Atxn1 in the brainstem. The two kinases are differentially expressed across brain regions in mice and humans, and they showed differential efficacy in regulating Atxn1 in a brain region-dependent manner. Our work demonstrates that a key phosphorylation site that impacts mutant ATXN1 levels can be modulated by different regulators in select brain regions, and show that targeting multiple regulators may expand therapeutic potential to rescue multiple degenerating brain areas. Results Genetic screen reveals that ZBTB7A and ZBTB7B regulate ATXN1, and ZBTB7B is the more potent regulator We discovered novel ATXN1 regulators by performing an shRNA screen of 858 genes in a Daoy (human brain cancer) cell line designed to report on ATXN1 levels (Fig 1A). This cell line expresses a mRFP-ATXN1(82Q)-IRES-YFP transgene where ATXN1 levels can be monitored by measuring the RFP/YFP fluorescence ratio (Park et al, 2013). After transducing the cell line with a pooled library of shRNAs (8,845 shRNAs, ~ 10 shRNAs/gene), cells displaying a decreased or increased RFP/YFP ratio (5% lowest and 5% highest fractions) were sorted and collected via fluorescence-activated cell sorting (FACS). Using the unsorted bulk population of cells as a reference, shRNAs enriched in the sorted fractions were identified by next-generation sequencing. Deconvoluting the genes targeted by these enriched shRNAs revealed potential ATXN1 regulators, which were grouped into either positive regulators (genes targeted by shRNAs in the 5% lowest) or negative regulators (genes targeted by shRNAs in the 5% highest). These regulators were filtered and ranked (see Materials and Methods) to identify 39 positive regulators and 13 negative regulators that when knocked down decrease or increase ATXN1 levels, respectively (Tables EV1 and EV2). Among the positive ATXN1 regulators, ZBTB7A and ZBTB7B, caught our attention as they are two close paralogs that ranked in the top five genes demonstrating a strong effect on ATXN1. Figure 1. Genetic screens reveal that ZBTB7A and ZBTB7B regulate ATXN1, and ZBTB7B shows more pronounced effect A diagram that outlines the steps of the genetic screen. ATXN1 reporter cell line produces RFP-conjugated ATXN1 and YFP internal control independently from a same transcript, and ATXN1 levels were monitored by measuring the ratio of red to yellow fluorescence intensity. (1) ATXN1 reporter cell line was transduced with lentivirus harboring shRNAs of 858 ubiquitination-related genes. (2, 3) The cells with the 5% lowest and 5% highest fluorescence ratio were sorted. (4) Illumina sequencing revealed enriched shRNAs in the two groups compared to the non-sorted group. (5) Identifying the genes targeted by these enriched shRNAs revealed 39 positive regulators including ZBTB7A and ZBTB7B, and 13 negative regulators of ATXN1 protein levels (See Table EV2 for the full list). Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/− (n = 4), Zbtb7b+/− (n = 3), Zbtb7a+/−; Zbtb7b+/− (n = 4) mice. Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm. Data information: (B–D) Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA. Source data are available online for this figure. Source Data for Figure 1 [embj2020106106-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint We first confirmed that knockdown of either ZBTB7A or ZBTB7B in the reporter cell line decreased ATXN1 using Western blot analysis (Fig EV1A). To test whether ZBTB7A and ZBTB7B regulate endogenous Atxn1 in neurons, we transduced primary cerebellar granule neurons (CGNs) with lentivirus containing shRNAs that target either gene. Knockdown of either Zbtb7a or Zbtb7b in CGNs decreased Atxn1 significantly (Figs 1B and EV1B), and simultaneous knockdown of both of them decreased Atxn1 to a greater extent than knockdown of either alone (Fig 1B). This was also true in the cerebella of mice where double heterozygous knockout of Zbtb7a and Zbtb7b decreased Atxn1 more than single heterozygous knockout of each gene (Fig 1C). Consistent with these results, overexpression of either Zbtb7a or Zbtb7b increased Atxn1 in the CGNs, and co-overexpression of both of them increased Atxn1 to a greater extent (Fig 1D). We next investigated the in vivo genetic interaction between ATXN1 and either ZBTB7A or ZBTB7B by expressing human ZBTB7A or ZBTB7B in the eyes of Drosophila also expressing human wild-type ATXN1(30Q) in the same region (Fernandez-Funez et al, 2000). These flies showed an increase of ATXN1 levels and with severe disruption of external ommatidia structures compared to the flies expressing ATXN1 without either ZBTB7A or ZBTB7B (Fig 1E). Altogether, these data indicate that ZBTB7A and ZBTB7B regulate ATXN1 positively and additively. Click here to expand this figure. Figure EV1. Genetic screen reveals that ZBTB7A and ZBTB7B regulate ATXN1, and ZBTB7B shows more pronounced effects Western blot analysis of ATXN1(82Q), ZBTB7A and ZBTB7B levels in ATXN1(82Q) reporter cell line after knockdown of either ZBTB7A or ZBTB7B (n = 2). Western blot analysis of Atxn1, Zbtb7a and Zbtb7b levels in cerebellar granule neurons (CGNs) after knockdown of either Zbtb7a or Zbtb7b (n = 3). Densitometry of Zbtb7a and Zbtb7b in the Western blot images from Fig 1B (left; n = 3) and Fig 1C (right; n = 3, 4, 3, 4, following the order of genotypes in the graph, respectively). Western blot analysis of Atxn1 levels after knockdown of either Zbtb7a or Zbtb7b and overexpression of the other (n = 3). Data information: (A–D) Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA. Source data are available online for this figure. Download figure Download PowerPoint Although both Zbtb7a and Zbtb7b regulate Atxn1 in vivo, there are remarkable differences between the two. While Zbtb7b−/− mice are viable (Wang et al, 2008b), Zbtb7a−/− mice are not (Maeda et al, 2009), suggesting that inhibiting Zbtb7b would be safer than Zbtb7a. Also, our in vitro and in vivo data show that Zbtb7b also had a greater effect on ATXN1 levels than Zbtb7a (Fig 1B–D). Notably, both knockdown and knockout of Zbtb7a induced a compensatory increase of Zbtb7b, but not vice versa (Figs 1B and C, and EV1, EV2, EV3, EV4, EV5, EV6), which might explain the weaker effect of Zbtb7a knockdown on Atxn1. To investigate the compensatory relationship between Zbtb7a and Zbtb7b in neurons, we measured Atxn1 levels after knocking down one of them and overexpressing the other simultaneously in CGNs. While Zbtb7a knockdown together with Zbtb7b overexpression did not change Atxn1 levels, Zbtb7b knockdown together with Zbtb7a overexpression did decrease Atxn1 protein levels, indicating that the Zbtb7a's role in regulating Atxn1 can be partially replaced with Zbtb7b, but not vice versa (Fig EV1D). From this one-way compensatory relationship between Zbtb7a and Zbtb7b, we concluded that Zbtb7b is the more potent ATXN1 regulator. ZBTB7B regulates ATXN1 indirectly through activating RSK3 expression To investigate the mechanisms underlying ATXN1 regulation by ZBTB7B, we first focused on the two distinct domains of ZBTB7B, the four ZF (Zinc Finger) domains which bind target DNA sequences (Klug, 2010), and the BTB (Broad-Complex, Tramtrack and Bric a brac) domain, which interacts with cullin3 E3 ligase (Xu et al, 2003) or transcriptional co-regulators (Perez-Torrado et al, 2006) (Fig 2A). With these two domains, ZBTB7B can either change gene expression or ubiquitinate proteins for degradation (Fig 2B). To determine which domain is involved in regulating ATXN1, both ZF and BTB domains were mutated within the full length ZBTB7B protein and overexpressed individually in Daoy cells, and ATXN1 levels were monitored. For ZF domain mutant (ZFmut), we generated an R363L mutation in the first ZF domain, because not only is R363 in direct contact with DNA (Pavletich & Pabo, 1991), but also mutating the corresponding residue of this in ZBTB7A (R399L) is known to abrogate target DNA binding ability (Liu et al, 2014). R49H and D35N/R49Q were used for BTB domain mutants (BTBmut) as they disrupt protein interacting function (Melnick et al, 2002) (Fig 2A). While the overexpression of BTBmut increased ATXN1 despite its low expression levels, overexpression of ZFmut did not, suggesting that DNA binding ZF domain is critical for regulating ATXN1 (Fig 2C). Therefore, we first hypothesized that ZBTB7B can change ATXN1 gene expression. However, the cerebella of Zbtb7b knockout mice showed no change in the Atxn1 mRNA levels (Fig 2D), suggesting that ZBTB7B indirectly regulates ATXN1 protein levels via unknown, intermediate regulators that can directly alter ATXN1 protein levels. To identify potential intermediate regulators, we performed an RNA-seq on RNA from Daoy cells expressing either the wild-type or ZFmut ZBTB7B, and analyzed differentially expressed genes (DEGs) (Fig 2E). The cells expressing WT ZBTB7B clearly showed an expression profile distinct from those expressing ZFmut ZBTB7B (Figs 2F and EV2A). After filtering our DEGs (see Materials and Methods), we focused on RSK3 (RPS6KA2) because its expression distinctively increase (4 fold) in cells overexpressing the wild-type ZBTB7B compared to those expressing ZFmut, and RSK3 could potentially regulate Atxn1 directly as it can post-translationally modify proteins in the nucleus where ATXN1 mainly resides (Fig 2E and Table EV3). Figure 2. ZBTB7B regulates ATXN1 indirectly through activating RSK3 expression ZBTB7B's domain structure marked with the mutations in BTB and ZF domain used in this study. Two representative biological functions of ZBTB7B: (1) changing target gene expression; (2) recruiting target proteins for ubiquitination. CUL3: Cullin3, E2: ubiquitin-conjugating enzyme, SUB: substrate. Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut), or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). qRT–PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/− (n = 4), and Zbtb7b−/− mice (n = 4). MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled. Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score. qRT–PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3
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