Rat Model of Cockayne Syndrome Neurological Disease

Report

Rat Model of Cockayne Syndrome Neurological Disease Graphical Abstract

Authors Yingying Xu, Zhenzhen Wu, Lingyun Liu, Jiena Liu, Yuming Wang

CSB Abnormal neurodevelopment

R571X

R571X

Correspondence [email protected]

In Brief

Impaired TC-NER UV light

CPD

5’

Dysmyelination

3’

3’

5’

Defective CPD Removal Healthy

CSBR571X/R571X

UV light

RNAP II 3’OH

Brain Atrophy

Failure of Transcription Restart

Highlights d

Modeling Cockayne syndrome in rat by mimicking the genetic defects of CS patients

d

CS rat model is impaired in TC-NER

d

d

Deficiency of CSB causes neurodevelopmental abnormalities in rat Aberrant gene expression is revealed in the cerebellar cortex of CS rats

Xu et al., 2019, Cell Reports 29, 800–809 October 22, 2019 ª 2019 The Author(s). https://doi.org/10.1016/j.celrep.2019.09.028

Xu et al. report a CRISPR/Cas9-edited rat displaying a nonsense mutation in the CSB gene, which can be used as an appropriate animal model for the DNA repair and neurodevelopment characteristics of Cockayne syndrome disease.

Cell Reports

Report Rat Model of Cockayne Syndrome Neurological Disease Yingying Xu,1,3 Zhenzhen Wu,1,3 Lingyun Liu,2,3 Jiena Liu,1 and Yuming Wang1,4,* 1Key Laboratory of Neurological Function and Health, School of Basic Medical Science, Guangzhou Medical University, Guangzhou 511436, China 2School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China 3These authors contributed equally 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.09.028

SUMMARY

Cockayne syndrome (CS) is a rare genetic neurodevelopmental disorder, characterized by a deficiency in transcription-coupled subpathway of nucleotide excision DNA repair (TC-NER). Mutation of the Cockayne syndrome B (CSB) gene affects basal transcription, which is considered a major cause of CS neurologic dysfunction. Here, we generate a rat model by mimicking a nonsense mutation in the CSB gene. In contrast to that of the Csb
/
mouse models, the brains of the CSB-deficient rats are more profoundly affected. The cerebellar cortex shows significant atrophy and dysmyelination. Aberrant foliation of the cerebellum and deformed hippocampus are visible. The white matter displays high glial fibrillary acidic protein (GFAP) staining indicative of reactive astrogliosis. RNA sequencing (RNA-seq) analysis reveals that CSB deficiency affects the expression of hundreds of genes, many of which are neuronal genes, suggesting that transcription dysregulation could contribute to the neurologic disease seen in the CSB rat models. INTRODUCTION Cockayne syndrome (CS) is a rare autosomal recessive disorder, mainly characterized by pronounced hypersensitivity to sunlight, severe growth failure, neurologic degeneration, and developmental abnormalities of multiple organ systems. CS patients can be divided into three subgroups according to the onset age and severity (Laugel, 2013). The major neuropathologic features of CS are a combination of mental retardation and profound brain atrophy; patchy demyelination in brain; striking calcification, mostly within the basal ganglia and cerebellum; notable brain vasculopathy; and degeneration of cerebellar Purkinje neurons (Brooks, 2013). CS is caused by mutations in the CSA and CSB genes, which encode for proteins involved in the transcription-coupled subpathway of nucleotide excision DNA repair (TC-NER) (Laugel et al., 2010). A diagnostic feature of CS is defective recovery of RNA synthesis in fibroblasts from patients after UV irradiation (Nakazawa et al., 2010).

However, the neurologic disease characteristics of human CS patients cannot be explained only by TC-NER deficiency. First, xeroderma pigmentosa (XP) patients with both TC-NER/globalgenome nucleotide excision repair (GG-NER) defects exhibit much milder clinical features than CS patients (Anttinen et al., 2008). Second, most neurodegenerative phenotypes (like cataracts and microcephaly, which are present prenatally) in CS do not support a causative role for accumulated DNA damage but are more consistent with a transcriptional defect resulting in increased susceptibility to ‘‘one-hit’’ degeneration (Clarke et al., 2000; Gorgels et al., 2007). It has also been proposed that the CSB protein is involved in base excision repair and oxidative DNA damage in mitochondria might be associated with CS neuropathology (Nardo et al., 2009; Scheibye-Knudsen et al., 2012). The precise molecular and cellular defects underlying CS neurologic disease are still poorly understood; one of the limitations to studies is that the current mutant mouse models that mimic the genetic defects of CS patients provide only a very limited opportunity to address these issues. Mice deficient for CSA or CSB reflect the TC-NER defect but fail to develop profound neurological abnormalities, although minor defects like age-dependent deafness and blindness have been reported before (Laposa et al., 2007; van der Horst et al., 1997). From a translational standpoint, the existing mouse models cannot be used to test potential therapeutics for CS neurologic disease. Here, we show that rats lacking expression of the full-length CSB protein display repair-deficient phenotypes and brain abnormalities, features that generally resemble symptoms observed in CS patients. This rat model may provide new insights into the neuropathologies of CS diseases.

RESULTS Generation of a CSB-Deficient Rat by CRISPR/Cas9 In CS patients, most mutations in the CSB gene are nonsense and frameshift mutations that are predicted to yield a truncated or degraded protein (Laugel et al., 2010). We chose to mimic the genotype R571X mutation (corresponding with the R652X mutation in at least two homozygous CSB patients diagnosed as type II CS and one allele in one compound-heterozygous CSB patient diagnosed as type I CS) (Figure 1A) (Calmels et al., 2018; Laugel et al., 2010). This position is highly conserved and resides within the ATPase domain of the CSB protein. We

800 Cell Reports 29, 800–809, October 22, 2019 ª 2019 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

A

R571X Ercc6 transcript 1 Ercc6 transcript 2

WT 5’...CAT AAA ATT CGA AAT CCA AAT GCT...3’ R571X 5’...CAT AAA ATT TGA AAT CCA AAT GCT...3’ (Stop)

B

rat Ercc6 locus 5’ ...GAAGGACATAAAATTCGAAATCCAAATGCTGCAGTCACCCTTGCTTGCAAACAGGTATAGCTG... 3’ 3’ ...CTTCCTGTATTTTAAGCTTTAGGTTTACGACGTCAGTGGGAACGAACGTTTGTCCATATCGAC... 5’ PAM sgRNA

ssODN template (s) 5’ ...GAAGGACATAAAATTTGAAATCCAAATGCTGCAGTCACGCTTGCTTGCAAACAGGTATAGCTG... 3’ R571X

1X

D

CSB R571X/+ CSB R571X/R571X

R

57

pe

180 kD

anti-α tubulin

AC A T A A A A T T T G A A A T C C A A A

SB

ty ild w WT

anti-CSB

AC A T A A A A T T C G A A A T C C A A A

1X

/R

57

AC A T A A A A T T C G A A A T C C A A A

C

C

50 kD

E #CHROM POS NC_005101. 204002779 NC_005103. 130886130 NC_005104. 143354741 NC_005104. 143354941 NC_005106. 6250092 NC_005106. 6250118 NC_005106. 78474733 NC_005106. 98974189 NC_005106. 98974249 NC_005106. 98974251 NC_005107. 36189 NC_005107. 53987436 NC_005113. 41173446 NC_005113. 107306087 NC_005114. 97639469 NC_005115. 8779715 NC_005115. 8779738 NC_005120. 105109799

Func REF ALT Mut1 DV_Mut1 DP_Mut1 Mut2 DV_Mut2 DP_Mut2 WT intergenic T C C/C 58 58 T/C 17 44 C/C intergenic T C C/C 50 50 C/C 37 37 C/C intergenic A T A/T 11 33 T/T 14 14 T/T intergenic C T C/T 32 67 C/C --C/C intergenic G A G/A 16 36 G/A 26 38 G/A intergenic A G A/G 15 39 A/G 23 38 A/G intergenic A G G/G 53 53 A/G 32 54 A/G intergenic G A A/A 43 43 A/A 47 47 A/A intergenic A G G/G 47 47 G/G 57 57 G/G intergenic T C C/C 48 48 C/C 57 57 C/C intergenic G A A/A 36 36 A/A 31 31 A/A intergenic C T C/T 27 45 T/T 36 37 C/C intergenic C G G/G 64 64 G/G 48 48 G/G intergenic A T T/T 39 39 A/T 20 42 T/T intergenic T C C/C 63 63 C/C 49 49 C/C exonic C T T/T 51 51 T/T 37 37 C/C exonic C G G/G 52 52 G/G 39 39 C/C intergenic T C T/T --T/T --C/C

DV_WT DP_WT 33 33 30 30 17 17 --21 38 18 36 22 35 36 36 36 36 35 35 18 18 --31 31 19 19 21 21 ----5 5

#CHROM, chromosome; POS, position of SNV (variant); REF, nucleotide in the reference genome; ALT, nucleotide alteration in the sample; DP, reads of sample being sequenced; DV, reads of SNV being sequenced.

Figure 1. Generation of the CSB Rat Model Expressing Mutant Ercc6 (R571X) Gene (A) Schematic illustration of CRISPR/Cas9-mediated generation of the CSB rat in which the Ercc6 gene was mutated at R571. The rat Ercc6 gene has two transcripts with the same DNA sequence flanking the targeting site. (B) A schematic representation showing the locations of the gRNA and single-strand oligodeoxynucleotide (ssODN). Blue letters indicate the position of the gRNA targets, with red letters highlighting the PAM sequences. The repair templates are shown at the bottom, with a red letter indicating the stop gain at R571. (C) Sanger sequencing shows the nonsense mutation in heterozygous and homozygous CSB rats. The black arrowheads indicate the mutated site. (D) Western blot of CSB protein in WT and CSBR571X/R571X rats. (E) Summary of off-target sites detected by whole-genome sequencing. R571X mutation is highlighted in blue; the mutation in the PAM sequence is presented in gray. Mut1 and Mut2 are two representives of the homozygous CSBR571X/R571X rats. See also Figure S1.

Cell Reports 29, 800–809, October 22, 2019 801

designed guide RNAs (gRNAs) targeting exon 8 of the rat CSB gene and donor template containing the R571X (CGA > TGA) mutation site. A silent mutation (ACC to ACG) was also introduced to the oligo donor to prevent the binding and re-cutting of the sequence by gRNAs after homology-directed repair (Figure 1B). We successfully generated an F0 rat line harboring the expected single-nucleotide substitution with a high efficiency of 10.2% (Figure S1A). Six homozygous CSBR571X/R571X rats were obtained and confirmed by genotyping (Figure 1C). RNA expression for CSB gene was relatively lower in CSBR571X/R571X rats than in either wild-type (WT) controls or the CSBR571X/+ littermates (Figures S1B and S1C), as consistent with what has been observed in the CS1AN cell line, which is derived from the CSB patient (Figure S1D). The full-length CSB protein was significantly diminished in the homozygous CSBR571X/R571X rats when compared with WT rats (Figure 1D), and no truncated form was detected (Figure S1E). Importantly, the CSBR571X/+ offspring were indistinguishable from WT rats in development, size, and behavior and were, therefore, used as a control throughout the study. Moreover, we used wholegenome sequencing (WGS) to comprehensively access onand off-target mutations in CSBR571X/R571X rats. We found that CRISPR/Cas9-based gene editing was active on the expected genomic sites without producing off-target modifications (single-nucleotide variants [SNVs] or indels) in other functional regions of the rat genome (Figure 1E). CSBR571X/R571X Rats Exhibit Repair Deficiency To test if disruption of the CSB gene in CSBR571X/R571X rats results in disturbed TC-NER, we analyzed several DNA repair parameters in primary rat fibroblasts from skin. First, CSBR571X/R571X rat fibroblasts were hypersensitive to exposure of increasing doses of UV when compared with the heterozygous CSBR571X/+ littermate controls (Figures 2A and S2A). The UVinduced cyclobutane pyrimidine dimers (CPDs) were rapidly removed in the control fibroblasts, and in contrast, as demonstrated in the Csb
/
mouse model (van der Horst et al., 1997), the CSBR571X/R571X fibroblasts displayed very low levels of repair at 48 h (Figure 2B), indicating defective repair at both the transcribed and the nontranscribed strands of genes in CSB-deficient cells. To investigate the effect of UV irradiation on transcription resumption, we measured the nascent RNA synthesis by 5-Ethynyl uridine (EU) immunolabeling. As Figure 2C shows, in response to UV, a distinct population of lowly transcribing cells was clearly detectable by 3 h; after 48 h, nascent RNA synthesis was gradually recovered in the CSBR571X/+ fibroblasts. Markedly, the percentage of lowly transcribed cells was increased in the CSBR571X/R571X fibroblasts by 48 h. This result was further confirmed by qRT-PCR, which revealed that RNA polymerase II (Pol II) failed to progress to the elongation phase in the cells derived from CSBR571X/R571X rats (Figure S2D). Next, we evaluated levels of DNA double-strand breaks (DSBs) by 0, 2, and 24 h after UV irradiation by detecting g-H2AX foci. In CSBR571X/+ cells, g-H2AX foci appeared most prominently at 2 h and decreased to almost the untreated level at 24 h. In CSBR571X/R571X cells, g-H2AX foci exhibited high levels over 24 h (Figure S2B). Previous studies have revealed that the GG-NER capacity of Csb
/
mouse fibroblasts was intact by

802 Cell Reports 29, 800–809, October 22, 2019

performing the unscheduled DNA synthesis (UDS) assay (van der Horst et al., 1997). Here, we used a non-radioactive technique by incorporating 5-Ethynyl-2’-deoxyuridine (EdU) to measure UDS, which has been shown to be sufficient for routine XP screening (Limsirichaikul et al., 2009). We found that both normal fibroblasts and CSB-deficient primary cells exhibited indistinguishable UDS activity after UV-C irradiation (Figure 2D), strongly indicating that CSBR571X/R571X rats are only compromised in the T cell receptor (TCR) pathway of NER. The less well-characterized function of CSB in the cellular response to oxidative DNA damage has been proposed to contribute to CS (de Waard et al., 2003; Tuo et al., 2001). We found that CSB-deficient rat fibroblasts were hypersensitive to potassium bromate relative to the CSBR571X/+ control (Figure S2C). Previous studies also proposed that mitochondrial impairments play an important role in CS (Berquist et al., 2012; Kamenisch and Berneburg, 2013; Scheibye-Knudsen et al., 2012). Here, we used qPCR to determine the levels of mtDNA and observed a significant reduction of mtDNA content in CSBR571X/R571X rats when compared with the controls (Figure S2E). More importantly, consistent with what has been detected in patient-derived CS cells (Chatre et al., 2015), expression of DNA polymerase POLG1 was decreased in CSBR571X/R571X fibroblasts (Figure S2F). From these data, we concluded that the CSB-deficient rat is defective in UV-induced DNA damage repair and accordingly forms an appropriate animal model for the repair characteristics of human CS disease. Brain Developmental Defects in CSBR571X/R571X Rats Although the overall appearance and behavior of CSBR571X/R571X rats seemed normal compared with CSBR571X/+ rats, the brain development exhibited notable abnormalities (see Table S1). As consistent with the cerebellar atrophy observed in CS patients’ post-mortem tissues (Figure S3A), H&E staining of sagittal sections from the 9-week-old CSBR571X/R571X rats revealed that the size of the cerebellum was much smaller compared with the control CSBR571X/+ littermates. The molecular layer and the granular layer of the cerebellar cortex were much thinner than that of the littermates (Figure 3A). Furthermore, foliation defects were also visible on sagittal sections, where the most anterior lobe did not form or the middle lobe was rudimentary (Figure 3B). Atrophy of the hippocampus is seldom reported in CSB patients. CSBR571X/R571X rats exhibited profound hippocampal dysplasia, including altered organization of cells in CA3 region and malformed dentate gyrus (Figure 3C). We stained Purkinje cells with anti-calbindin antibody and found that aberrant position of Purkinje cell somas comprised the most obvious lesion in CSBR571X/R571X rats (Figure 3D), consistent with the clinical studies showing that degeneration of Purkinje neurons is the major feature of CS neurologic disease (Brooks, 2013). Microtubule structures were also affected in the 9-week-old CSBR571X/R571X rats, with decreased neurofilament expression (Figure 3E), indicating severe axonal degeneration. Global patchy demyelination has been most well documented in post-mortem studies of CS patients, and MRI of CS patients also concluded that CS is primarily a hypomyelination disease (Koob et al., 2010). Crucially, some segments of axons in the white matter of the cerebellum lacked myelination in the 9-week-old CSBR571X/R571X rats

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Figure 2. Impaired TC-NER and Intact GG-NER of CSBR571X/R571X Rat Fibroblasts from Skin in Response to UV Irradiation (A) Cell viability of CSBR571X/+ and CSBR571X/R571X cells after 3 days of treatment with the indicated dose of UV-C irradiation. Error bars represent means ± SD; n = 3 independent experiments for each cell line.

(legend continued on next page)

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compared with controls (Figure 3F). Notably, this has not been detected in Csb
/
mouse models. However, in Csb
/
mouse models, an increased frequency of activated microglial cells and astrocytosis within the white matter was observed (Jaarsma et al., 2011). Therefore, we characterized the phenotype of astrocytes by testing GFAP expression. Notably, we detected substantial astrocyte activation in the CSBR571X/R571X rat cerebella (Figure 3G). Brain calcification in CS has been reported to occur bilaterally and symmetrically (Koob et al., 2010); nonetheless, histological examination of cerebra in CSBR571X/R571X rats failed to reveal obvious abnormalities (Figures S3B and S3C). Neither the small crystals of calcium nor the expression of collagen IV protein around brain vascular elements was detected (Zarb et al., 2019). Our results suggest that neurodevelopmental abnormalities take place in CSBR571X/R571X rats and are associated with statistically significant differences illustrated in the form of GFAP upregulation compared with the control (see the comparisons of neurologic diseases between CS patients and animal models in Table S1). Mechanistic Insights on Brain Abnormalities in CSBR571X/R571X Rats Because it has been proposed that the important role of CSB for defective transcription regulation can provide a better explanation for many aspects of CS neurologic characteristics than defective DNA repair (Bailey et al., 2012; Bradsher et al., 2002; Newman et al., 2006; Wang et al., 2016), we performed RNA sequencing to determine whether global gene expression was dysregulated in CSBR571X/R571X rats. RNA was isolated from cerebellar cortex and then subjected to RNA sequencing analysis. Gratifyingly, bioinformatic analysis of the gene expression signatures showed that all the samples derived from six CSBR571X/R571X rats clustered together and separated from the WT controls and the CSBR571X/+ littermates (Figure 4A). A total of 378 genes (221 downregulated and 157 upregulated) were dysregulated in the CSBR571X/R571X rat cerebella compared with that of the control littermates (Table S2), among which a significant proportion are also affected in post-mortem CS brain tissues (Figure 4D). Gene Ontology (GO) term enrichment analysis revealed that genes involved in neuronal function and ion channel activity were significantly upregulated in the CSBR571X/R571X samples (Figure 4B), which is consistent with the neurologic diseases observed in the animal model. Interestingly, when we manually interrogated our RNA sequencing (RNA-seq) data, we found that genes encoding components of the voltage-gated potassium channels (including KCND3, KCNF1, and KCNAB1) were among the most upregulated genes (Figure 4C). It has been reported that dysregulation

of voltage-gated potassium channels can result in degeneration of the cerebellum (Lee et al., 2012). Notably, genes involved in exocytosis, such as synaptic SNAREs (SNAP25), dense core granules (SYT10), and synaptic vesicle fusion (RAB3A), were also significantly deregulated in CSBR571X/R571X rats. In our previous studies, we demonstrated that a widespread impairment of regulated secretion induced by abnormal expression of these proteins is highly likely associated with multiple neurodevelopmental defects observed in CS patients (Wang et al., 2014, 2016). It is particularly noteworthy that among the dysregulated genes, several genes are crucial for neuronal development and function. For example, TUBB3 and TUBB4A, which are the major constituent of microtubules, play a critical role in axon guidance and maintenance (Tischfield et al., 2010). Neurod1 transcription activator is required for neuronal differentiation and function in cerebellar cortex (Cho and Tsai, 2004). Also, the ephrin receptor has been implicated in mediating developmental events in the central nervous system (Yue et al., 1999). Taken together, our results demonstrate that the loss of CSB function in the CSBR571X/R571X rat model exhibits a prominent defect on the expression of neuronal genes that may contribute to the neurologic characteristics observed in this animal model. DISCUSSION There are many cognitive and physiological characteristics that make the rat a better choice than the mouse for laboratory studies in neurobiology, cardiobiology, and immunology (Abbott, 2004). In addition, rat models are superior to mouse models for testing the pharmacodynamics and toxicity of potential therapeutic compounds, particularly because of their larger size and their similar detoxifying enzymes to those in humans (LindbladToh, 2004). Here, we generated a mutant rat with a substantial defect in TC-NER to establish an experimental animal model for the NER disorder, which can complement the existing CS mouse models. Previous studies have demonstrated that reduced lifespan and severe neurologic dysfunctioning were not observed in Csb
/
mice, except very mild growth disturbance, like deafness and vision loss (van der Horst et al., 1997). In this study, we have found that CSBR571X/R571X rats exhibit neurological symptoms that resemble those of CSB patients significantly more than mouse models. In severe CS cases, the cerebellum is already very small at the time of first evaluation (at birth in some cases), indicative of very early cerebellar hypoplasia (Koob et al., 2010) (Figure S3A). In CSBR571X/R571X rats, the size of the cerebellum was strikingly smaller than that of the control CSBR571X/+ littermates, including significant atrophy of both the molecular layer and the granule layer. Notably, the volume (of Purkinje neurons)

(B) CPD immunostaining of UV-irradiated (16J/m2) CSBR571X/R571X and CSBR571X/+ rat fibroblasts. Scale bar represents 50 mm. Average CPD intensity/nuclei is shown as a histogram plot. n = 500 cells from three biological replicates. (C) Nascent RNA transcription was accessed by EU staining in the absence and presence of UV irradiation (16J/m2). Scale bar represents 50 mm. Histogram plots showing average EU incorporation. n = 500 cells from three biological replicates. (D) Representative images of the EdU incorporation are shown. Scale bar represents 50 mm. White arrows indicate non-S-phase cells with unscheduled DNA synthesis (UDS) occurring. UDS levels were normalized and expressed as percentages of the cells with UDS in the total cell number as shown in the bar chart. Error bars represent means ± SD. Student’s t test; n = 200 cells from three biological experiments. See also Figure S2.

804 Cell Reports 29, 800–809, October 22, 2019

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CSBR571X/R571X

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CSBR571X/R571X #5

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cortex length (μm)

600

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450 300 150

2mm

57 R

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Cell Reports 29, 800–809, October 22, 2019 805

and the structure (of folia) of the cerebellum were also disturbed. More importantly, we saw no dramatic increase in apoptosis in either the cerebella or the cerebra of CSBR571X/R571X rats (no stimulation of gH2AX expression was observed by immunofluorescence; Figure S3D), suggesting that the loss of CSB function did not render the neuronal cells vulnerable to cell death resulting from DNA damage accumulation. It has been speculated before that early microcephaly and cerebellar hypoplasia seen in CS neurologic disease are developmental abnormalities that are fundamentally different than aging (Brooks, 2013). Our current studies in the rat model clearly suggest that the mechanism contributing to cerebellar pathology is not solely DNA repair deficiencies. Likewise, the GFAP-positive cells detected in CSBR571X/R571X rats were already present at 9 weeks of age. In terms of the mechanistic basis of CS neurologic disease, many researchers proposed a transcription syndrome hypothesis (Brooks et al., 2008; Compe and Egly, 2012; Hoeijmakers et al., 1996; van Gool et al., 1997). A particularly important finding in support of it was that of Weiner and colleagues who used a microarray approach to investigate the effects of CSB on gene expression (Newman et al., 2006). They suggested that alterations of the CSB protein were unable to maintain chromatin structure in the basal state, which highly likely contributed to the abnormalities of growth-related characteristics. In our previous studies, we also found that Pol II transcription defects resulted in impaired neuritogenesis in CSB-depleted cells in the absence of external DNA damage (Wang et al., 2014, 2016). Not only limited to the clinical features of CS, studies on mouse models of CS also implied the expanded transcription hypothesis as a better explanation for the complex neuropathology. Jaarsma et al. (2011) has shown that activated microglia in Csb
/
mouse models were already present at 10 weeks of age, and the number did not increase with age, as would have been expected from an accumulated DNA damage hypothesis. Cleaver’s group has shown that in their Csb
/
mice, a number of genes relevant to neurobiology, transcription, and oxidative stress showed significantly altered expression compared with WT littermate controls. They also identified six overexpressed neural genes, which include myelin basic protein, synaptogyrin 3, neogenin, and the potassium voltage-gated channel, shaker-related subfamily, beta member 1 (Laposa et al., 2007).

In our study, we also found that neuronal genes are upregulated in CSBR571X/R571X rats, particularly genes critical for neuron cell differentiation and function, including NeuroD1, TUBB3, and SYT10. CS is a devastating neurodevelopmental disorder; however, the development of rational therapeutic strategies based on the understanding of the fundamental mechanistic characteristics of CS neurologic disease is largely lacking. The possibility that transcription dysregulation is a major cause of CS is ready to be translated into a therapeutic strategy because a more consistent pattern of transcription abnormality has been identified by us and others (Brooks, 2013; Newman et al., 2006; Wang et al., 2014, 2016). In our previous studies, we proposed that the neuron growth factor brain-derived neurotrophic factor (BDNF) could bypass the function of CSB in rescuing partial neuritogenesis defect in cells (Wang et al., 2016). In this study, among the affected genes identified in CSBR571X/R571X rats, NeuroD1 is particularly worthy of further investigation as a potential therapeutic target because it has been found that NeuroD1 and BDNF are coordinately expressed during neural development (Chatterjee et al., 2013). STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Rats B Primary rat fibroblasts B Human samples METHOD DETAILS R571X/R571X B Generation of CSB rats B Isolation of primary rat skin fibroblasts B Identification of off-target sites by whole genome sequencing B Clonogenic survival assay B Nascent RNA synthesis evaluation B 50 Ethynyl Uridine Staining

Figure 3. Histological Abnormalities in Brains of 9-Week-Old CSBR571X/R571X Rats (A) The overall length of the cerebellar cortex was shorter in 9-week-old CSBR571X/R571X rats compared with that of CSBR571X/+ rats. *p < 0.05 (Student’s t test; n = 6 rats). Data are expressed as means ± SD for six biological replicates with three technical replicates for each. (B) Cerebellar foliation defects in CSBR571X/R571X rats. Deformation (red box) and loss (black arrows) of folia can be seen in CSB mutant rats. Scale bar represents 1 mm. Representative images of six biological replicates are shown. (C) Abnormal development of the hippocampus in the CSB mutant rats. H&E staining of hippocampal sections of 9-week-old CSBR571X/R571X rats shows the morphologically distinct dentate gyrus (black box) and the disorganized appearance of the CA3 region compared with the control rats. Scale bar represents 100 mm. Representative images of six biological replicates are shown. (D) Abnormal density of Purkinje cells in CSBR571X/R571X rats. Cerebellar sections were stained with antibodies against calbindin. Arrows indicate Purkinje cells. Scale bar represents 50 mm. Representative images of six biological replicates are shown. (E) Immunofluorescence of the cerebellum of CSBR571X/R571X rat and the control rat labeled with anti-neurofilament antibody to visualize axons. Note that in the white matter regions of the cerebella (white box), neurofilament expression is much less in the CSB mutant rat. Scale bar represents 100 mm. Representative images of six biological replicates are shown. (F) Immunofluorescence of the cerebellum of CSBR571X/R571X rat and the control rat labeled with anti-MBP antibody to visualize myelination in the white matter. Arrows indicate dysmyelination in CSBR571X/R571X rats. Scale bar represents 50 mm. Representative images of six biological replicates are shown. (G) Representative images of immunohistochemistry of rat cerebellar cortex with GFAP (brown) and neuronal nuclei staining (blue). Quantification of GFAP cells per mm2 is shown. Student’s t test; n = 6 rats with three technical replicates for each. Arrows indicate microglial cells. See also Figure S3 and Table S1.

806 Cell Reports 29, 800–809, October 22, 2019

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CSBR571X/R571X_3

CSBR571X/R571X_2

CSBR571X/R571X_1

WT-3

WT-2

WT-1

CSBR571X/+_2

CSBR571X/+_1 GO Terms

B passive transmembrane transporter activity channel activity gated channel activity voltage-gated potassium channel activity inorganic cation transmembrane transporter activity substrate-specific channel activity ion channel activity metal ion transmembrane transporter activity monvalent inorganic cation transmembrane transporter ... potassium ion transmembrane transporter activity transporter complex myelin sheath transmembrane transporter complex axon plasma membrane region neuronal cell body presynapse somatodendritic compartment synapse synapse part inorganic cation transmembrane transport positive regulation of ion transmembrane ... cation transmembrane transport inorganic ion transmembrane transport response to ammonium ion monovalent inorganic cation transport positive regulation of cation transmembrane ... positive regulation of calcium ion transport neurotransmitter transport positive regulation of calcium ion ... 0.05

Count 6 8 10 12

padj 1.00 0.75 0.50 0.25 0.00

0.075 0.100 0.125

Gene Ratio

C

D 0

1

2

gabrg2 snap25 penk syt10 efnb2 tubb3 rab3a drd1 tubb4a neurod1 gdap1 kcnd3 kcnf1 kctd4 kcnab1 Padj 0.06

3

DEG in CS patients

4 Log2FC

DEG in CSB rats 200

0.04

0.02

21

1302

0

Figure 4. RNA-Seq Analysis of CSBR571X/R571X Rat Cerebella (A) Dendrogram shows hierarchical unsupervised clustering analysis of gene expression signatures from CSBR571X/R571X, WT, and CSBR571X/+ rat cerebella. (B) Top 30 enriched GO terms for differentially expressed genes in CSBR571X/R571X rats. Low adjusted p values (padjs) are in red and high padj values are in blue. The size of the circle (count) is proportional to the number of enriched genes in each representative GO term. Gene ratio means the ratio of the number of genes classified in the indicated GO categories to the number of all differentially expressed genes. (C) Examples of specific dysregulated genes with their respective fold change (Log2FC) differences and associated p values (padj). (D) Venn diagram shows the unique and overlapping differentially expressed genes in CSB rats and CSB patients. p = 0.005633502. See also Table S2.

Cell Reports 29, 800–809, October 22, 2019 807

B

EdU staining Immunofluorescence B Immunolabeling of CPD B Western blotting B Immunohistochemistry B RNA sequencing QUANTIFICATION AND STATISTICAL ANALYSIS B DEG analysis B Functional enrichment analysis DATA AND CODE AVAILABILITY B

d

d

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.09.028. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant number 81571092) and the Guangzhou Medical University Startup (grant number B195002002044) to Y.W. We thank Novogene sequencing facility for their expert assistance. Michelle Harreman is thanked for comments. AUTHOR CONTRIBUTIONS L.L. and Y.W. conceived and supervised the project. Y.X. and Z.W. performed all experiments, although J.L. was responsible for the western blot. Y.W. wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: February 4, 2019 Revised: August 26, 2019 Accepted: September 11, 2019 Published: October 22, 2019 REFERENCES Abbott, A. (2004). Laboratory animals: the Renaissance rat. Nature 428, 464–466. Anttinen, A., Koulu, L., Nikoskelainen, E., Portin, R., Kurki, T., Erkinjuntti, M., Jaspers, N.G., Raams, A., Green, M.H., Lehmann, A.R., et al. (2008). Neurological symptoms and natural course of xeroderma pigmentosum. Brain 131, 1979–1989. Bailey, A.D., Gray, L.T., Pavelitz, T., Newman, J.C., Horibata, K., Tanaka, K., and Weiner, A.M. (2012). The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferonlike and innate antiviral responses in CSB-null cells. DNA Repair (Amst.) 11, 488–501. Berquist, B.R., Canugovi, C., Sykora, P., Wilson, D.M., III, and Bohr, V.A. (2012). Human Cockayne syndrome B protein reciprocally communicates with mitochondrial proteins and promotes transcriptional elongation. Nucleic Acids Res. 40, 8392–8405. Bradsher, J., Auriol, J., Proietti de Santis, L., Iben, S., Vonesch, J.L., Grummt, I., and Egly, J.M. (2002). CSB is a component of RNA pol I transcription. Mol. Cell 10, 819–829. Brooks, P.J. (2013). Blinded by the UV light: how the focus on transcriptioncoupled NER has distracted from understanding the mechanisms of Cockayne syndrome neurologic disease. DNA Repair (Amst.) 12, 656–671. Brooks, P.J., Cheng, T.F., and Cooper, L. (2008). Do all of the neurologic diseases in patients with DNA repair gene mutations result from the accumulation of DNA damage? DNA Repair (Amst.) 7, 834–848.

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Nakazawa, Y., Yamashita, S., Lehmann, A.R., and Ogi, T. (2010). A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives. DNA Repair (Amst.) 9, 506–516. Nardo, T., Oneda, R., Spivak, G., Vaz, B., Mortier, L., Thomas, P., Orioli, D., Laugel, V., Stary, A., Hanawalt, P.C., et al. (2009). A UV-sensitive syndrome patient with a specific CSA mutation reveals separable roles for CSA in response to UV and oxidative DNA damage. Proc. Natl. Acad. Sci. USA 106, 6209–6214. Newman, J.C., Bailey, A.D., and Weiner, A.M. (2006). Cockayne syndrome group B protein (CSB) plays a general role in chromatin maintenance and remodeling. Proc. Natl. Acad. Sci. USA 103, 9613–9618. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281– 2308. Scheibye-Knudsen, M., Ramamoorthy, M., Sykora, P., Maynard, S., Lin, P.C., Minor, R.K., Wilson, D.M., III, Cooper, M., Spencer, R., de Cabo, R., et al. (2012). Cockayne syndrome group B protein prevents the accumulation of damaged mitochondria by promoting mitochondrial autophagy. J. Exp. Med. 209, 855–869. Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. Tischfield, M.A., Baris, H.N., Wu, C., Rudolph, G., Van Maldergem, L., He, W., Chan, W.M., Andrews, C., Demer, J.L., Robertson, R.L., et al. (2010). Human

van Gool, A.J., van der Horst, G.T., Citterio, E., and Hoeijmakers, J.H. (1997). Cockayne syndrome: defective repair of transcription? EMBO J. 16, 4155– 4162. van der Horst, G.T.J., van Steeg, H., Berg, R.J.W., van Gool, A.J., de Wit, J., Weeda, G., Morreau, H., Beems, R.B., van Kreijl, C.F., de Gruijl, F.R., et al. (1997). Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition. Cell 89, 425–435. Wang, Y., Chakravarty, P., Ranes, M., Kelly, G., Brooks, P.J., Neilan, E., Stewart, A., Schiavo, G., and Svejstrup, J.Q. (2014). Dysregulation of gene expression as a cause of Cockayne syndrome neurological disease. Proc. Natl. Acad. Sci. USA 111, 14454–14459. Wang, Y., Jones-Tabah, J., Chakravarty, P., Stewart, A., Muotri, A., Laposa, R.R., and Svejstrup, J.Q. (2016). Pharmacological Bypass of Cockayne Syndrome B Function in Neuronal Differentiation. Cell Rep. 14, 2554–2561. Yue, Y., Widmer, D.A., Halladay, A.K., Cerretti, D.P., Wagner, G.C., Dreyer, J.L., and Zhou, R. (1999). Specification of distinct dopaminergic neural pathways: roles of the Eph family receptor EphB1 and ligand ephrin-B2. J. Neurosci. 19, 2090–2101. Zarb, Y., Weber-Stadlbauer, U., Kirschenbaum, D., Kindler, D.R., Richetto, J., Keller, D., Rademakers, R., Dickson, D.W., Pasch, A., Byzova, T., et al. (2019). Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response. Brain 142, 885–902.

Cell Reports 29, 800–809, October 22, 2019 809

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-g H2AX

Abcam

Cat#ab22551; RRID:AB_447150

Mouse monoclonal anti-CPD

Cosmo Bio Co

Cat#CAC-NM-DND-001; RRID:AB_1962813

Rabbit polyclonal anti-CSB

Abcam

Cat#ab217202

Rabbit monoclonal anti-Calbindin

Abcam

Cat#ab108404; RRID:AB_10861236

Rabbit polyclonal anti-Neurofilament

Abcam

Cat#ab8135; RRID:AB_306298

Mouse monoclonal anti-MBP

Abcam

Cat#ab62631; RRID:AB_956157

Rabbit polyclonal anti-Collagen IV

Abcam

Cat#ab6586; RRID:AB_305584

Rabbit polyclonal anti-GFAP

Abcam

Cat#ab7260; RRID:AB_305808

Anti-rabbit HRP

Jackson ImmunoResearch

Cat#711-035-152; RRID:AB_10015282

Mouse Alexa Fluor PLUS 488

ThermoFisher Scientific

Cat#A32723; RRID:AB_2633275

Rabbit Alexa Fluor PLUS 488

ThermoFisher Scientific

Cat#A32731; RRID:AB_2633280

Mouse Alexa Fluor PLUS 647

ThermoFisher Scientific

Cat#A32728; RRID:AB_2633277

Rabbit Alexa Fluor PLUS 647

ThermoFisher Scientific

Cat#A32733; RRID:AB_2633282

Formalin-fixed postmortem brain tissue for CS case

NICHD Brain and Tissue Bank for Developmental Disorders

Case ID: 1762

Formalin-fixed postmortem brain tissue for CS case

NICHD Brain and Tissue Bank for Developmental Disorders

Case ID: 1920

Formalin-fixed postmortem brain tissue for control case

NICHD Brain and Tissue Bank for Developmental Disorders

Case ID: 1284

Formalin-fixed postmortem brain tissue for control case

NICHD Brain and Tissue Bank for Developmental Disorders

Case ID: 1500

Antibodies

Biological Samples

Chemicals, Peptides, and Recombinant Proteins Potassium Bromate

Sigma-Aldrich

309087

iQ SYBR green supermix

Bio-Rad

1708880

Critical Commercial Assays AllPrep DNA/RNA Mini Kit

QIAGEN

80204

RNeasy Mini Kit

QIAGEN

74106

RNeasy Lipid Tissue Mini Kit

QIAGEN

74804

TaqMan Reverse Transcription Reagents

ThermoFisher Scientific

N8080234

Click-iT RNA Alexa Fluor 488 Imaging Kit

ThermoFisher Scientific

C10329

Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye

ThermoFisher Scientific

C10337

TruSeq RNA Library Preparation Kit v2

Illumina

RS-122-2001

HiSeq PE Rapid Cluster Kit v2

Illumina

PE-402-4002

TruSeq Nano DNA High Throughput Library Prep Kit

Illumina

20015965

Deposited Data Whole genome sequencing

This manuscript

SRA: PRJNA550214

RNA-sequencing

This manuscript

GEO: GSE126032

Experimental Models: Cell Lines CSBR571X/R571X rat primary fibroblasts

This manuscript

N/A

CSBR571X/+ rat primary fibroblasts

This manuscript

N/A

WT SD rat primary fibroblasts

This manuscript

N/A (Continued on next page)

e1 Cell Reports 29, 800–809.e1–e5, October 22, 2019

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rat: CSBR571X/R571X

Cyagen Biosciences, Laboratory of China

This manuscript

Rat: CSBR571X/+

Cyagen Biosciences, Laboratory of China

This manuscript

Rat: Sprague Dawley (SD)

Cyagen Biosciences, Laboratory of China

This manuscript

This manuscript

N/A

Experimental Models: Organisms/Strains

Oligonucleotides All oligonucleotides used in this study are listed in Table S3 Software and Algorithms BWA

Li and Durbin, 2009

http://maq.sourceforge.net/

SAMtools

Li et al., 2009

http://samtools.sourceforge.net/

Picard

https://sourceforge.net/projects/picard/

N/A

seqtk

https://tracker.debian.org/pkg/seqtk

N/A

HISAT 2

Kim et al., 2015

http://ccb.jhu.edu/software/hisat/index.shtml

String Tie

http://ccb.jhu.edu/software/stringtie/

N/A

edgeR

http://www.bioconductor.org/packages/ release/bioc/html/edgeR.html

N/A

GraphPad Prism 6

GraphPad

https://www.graphpad.com/

ImageJ

Schneider et al., 2012

https://imagej.nih.gov/ij/

DMEM/F12

ThermoFisher Scientific

11320082

Antibiotic-Antimycotic (100X)

ThermoFisher Scientific

15240112

Liberase Blendzyme 3

Roche

11814176001

Other

Fetal Bovine Serum

Corning

35-010-CV

VECTASHIELD Antifade Mounting Medium containing DAPI

Vector Laboratories

H-1200

Formaldehyde solution

Sigma-Aldrich

252549

Triton X-100

Sigma-Aldrich

282103

Paraformaldehyde

Sigma-Aldrich

158127

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yuming Wang ([email protected]). Rat lines generated in this study have been deposited to Cyagen Biosciences, Laboratory of China (contract number: KICRS161107AJ1) and are available on request without restriction. EXPERIMENTAL MODEL AND SUBJECT DETAILS Rats All rats were generated on a pure SD background or were fully backcrossed to the SD background. All rats were bred and maintained under specific pathogen free conditions in accordance with the animal care and use regulations of the Cyagen Biosciences. Rats for experiments were age-matched (9-week old rats were obtained for all experiments), were littermates when possible, and were assigned to experimental groups based on genotype. Brains were removed and appropriate regional dissections performed before freezing the samples in liquid nitrogen. All experiments with animals were approved by the Animal Ethics Committee of Guangzhou Medical University. Primary rat fibroblasts The primary rat fibroblasts derived from the homozygous CSBR571X/R571X rats and the heterozygous CSBR571X/+ controls were generated in this study. All the cells were cultured in DMEM/F12 media plus 15% fetal bovine serum (FBS) and 1 X antibiotic/antimycotic. All cultures were routinely maintained at 37 C in a humidified 5% CO2 incubator.

Cell Reports 29, 800–809.e1–e5, October 22, 2019 e2

Human samples Patient-derived brain tissues were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders. Use of human tissue for post-mortem studies was reviewed and approved by the NICHD Brain and Tissue Bank ethics committee. METHOD DETAILS Generation of CSBR571X/R571X rats CSBR571X/R571X rats harboring the desired point mutation were generated on the SD. Rat background by Cyagen Biosciences using CRISPR/Cas9-based targeting and homology-directed repair. One pair of sgRNAs targeting the rat CSB locus (GenBank accession number: NM_001107296.1; Ensembl: ENSRNOG00000030017) was designed using online software: http://zifit.partners.org/ZiFiT/ Disclaimer.aspx. Oligonucleotides coding for the sgRNAs were annealed and assembled with a pX335 vector (Addgene) using the method described by Zhang at the Broad Institute of MIT (Ran et al., 2013). The oligo donor for homology directed repair (HDR) at the CSB gene was designed to introduce an in-frame amino acid substitution (R571X. CGA > TGA). A silent mutation (ACC to ACG) was also introduced to prevent the binding and re-cutting of the sequence by gRNA after HDR. The nickase expression vector containing the sgRNAs was transcribed in vitro. The resulting mRNA was artificially capped and polyadenylated to facilitate its proper translation into protein in mammalian cells. The in vitro transcribed nickase mRNA and oligo donor carrying the desired mutations were co-injected into fertilized rat eggs, followed by implantation of the eggs into surrogate mothers to obtain offspring. A total of 210 live eggs (SD rat strain) were injected. The site targeted by the nickase was PCR-amplified from pups, followed by sequencing of the PCR product to identify founder rats. A total of 3 founders were crossed with wild-type rats of SD background to generate heterozygous CSBR571X/+ rats, that were then intercrossed to give homozygous CSBR571X/R571X rats. The lack of mutations in off-target regions was verified by whole genome sequencing. Isolation of primary rat skin fibroblasts Rats were anesthetized with aether, the underarm area was cleaned with 70% ethanol and allowed to dry. Approximately 1 cm2 fragment of the skin was excised and placed immediately in sterile PBS to avoid drying. The tissue was cut into 1 mm pieces until the skin resembled putty, transferred into a sterile 30 mL beaker containing 10 mL of DMEM/F12, 0.14 Wunsch units/mL liberase blendzyme 3 and 1 X antibiotic/antimycotic, incubated at 37 C for 30-90 minutes with stirring. After digestion, the tissue fragments were rinsed 3 times with 10 mL of warm DMEM/F12 media containing 15% FBS and 1 X antibiotic/antimycotic, and then centrifuged at 524 X g. The pellet was resuspended in 10 mL of warm DMEM/F12 media with 15% FBS, 1 X antibiotic/antimycotic. The tissue pieces were broken down by pipetting with maximum force. Another 30 mL of DMEM/F12 media with 15% FBS, 1 X antibiotic/antimycotic was added and the solution was centrifuged twice at 524 X g to ensure removal of Liberase. The cell pellets were resuspended in 10 mL of DMEM/F12 media with 15% FBS, 1 X antibiotic/antimycotic and transferred to a 10 cm tissue culture dish and placed in a tissue culture incubator at 37C, 5% CO2. Identification of off-target sites by whole genome sequencing Genomic DNA was extracted from cerebellar cortex with AllPrep DNA/RNA mini kit (QIAGEN) according to the manufactory’s instructions and subjected to standard whole-genome DNA-library preparation for high-throughput sequencing (illumina platform) with a mean coverage of 60 X for mutant samples and 30 X for the wild-type sample. Valid and high-quality sequencing data was aligned to the reference genome of Rattus norvegicus (https://www.ncbi.nlm.nih.gov/genome/73?genome_assembly_id=203777) by BWA, and results were ranked using a comparison of SAMtools. Finally, duplicate reads were marked using the Picard. All polymorphic SNVs and indel sites in the rat genome were extracted, and high confidence SNVs and indel datasets were obtained and analyzed. Obtained datasets were used to compare the sgRNA sequence with the reference genome to get highly confident SNVs and indels. These off-target sites contained 1-5 base mismatches with the 20-bp target sequence and at least one mismatch in the PAM-proximal seed region via NCBI alignment tool blastn. To find more SNVs and indels sites, sgRNA homologous regions were amplified 100 bp in their upstream and downstream, respectively. All SNVs and indels were further filtered according to identity with reference and WT control. Clonogenic survival assay Exponentially growing primary fibroblasts were plated in 6-well tissue culture plates (1 X 104 cells). After incubation for 24 h, the cells were exposed to UV-C irradiation form 0-20 J/m2, and the cultures were maintained until surviving cells formed colonies (2 weeks). For KBrO3 treatment, cells were treated with different concentrations of KBrO3 for 3 h and maintained in fresh medium. Cells were fixed and stained with 0.5% crystal violet in absolute methanol for 15 min. Images were captured with Bio-Rad ChemiDoc imaging systems. Colonies from three biological replicates (each seeded into triplicate wells) were counted. Nascent RNA synthesis evaluation Total RNA was extracted from cultured primary skin fibroblasts with an RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions. The integrity of the RNA was tested on a denaturing agarose gel. RNA quality and quantity were also assessed with a

e3 Cell Reports 29, 800–809.e1–e5, October 22, 2019

Nanodrop spectrophotometer (ThermoFisher Scientific). For quantitative RT-PCR (qRT-PCR) analysis, single-stranded cDNA was synthesized from 200 ng of total RNA using a TaqMan Reverse Transcription Kit (Invitrogen). 50 Ethynyl Uridine Staining EU staining to detect newly synthesized RNA was performed according to the manufacturer’s instructions (Click-iT RNA imaging Kits, Invitrogen). Primary fibroblasts were exposed to 16 J/m2 UV-C irradiation and incubated for the indicated period of time. Media was replaced with fresh media containing 0.75 mM 50 Ethynyl uridine (EU) and cells were incubated for 2 h. EU-containing media was then removed and cells were fixed in PBS buffered formaldehyde (3.7%) for 45 min at room temperature, washed once with PBS and followed by permeabilization with 0.5% Triton X-100 diluted in PBS for 30 min. Cells were washed once with PBS and then Alexa Fluor 488 Azide fluorophores were covalently attached to EU-containing RNA by click reaction for 1 h at room temperature. Cells were then counterstained and mounted with mounting medium containing DAPI. Automated image acquisition of at least 5 fields per well was performed (Leica, TCS SP8). EdU staining Primary fibroblasts were cultured on coverslips and maintained at confluent density. Cells were washed with PBS once, followed by irradiation with 16 J/m2 UV-C. After UV irradiation, cells were immediately incubated with serum-free fresh medium supplemented with 10 mM EdU for 2 h. Cells were then washed with PBS once, followed by fixation with 3.7% formaldehyde for 15 minutes and permeabilization with PBS containing 0.5% Triton X-100 for 20 minutes. After extensive washing with PBS, cells were blocked with 10% FBS in PBS for 30 min. Incorporated EdU was detected by fluorescent-azide coupling reaction. Photographs of the cells were captured with a confocal microscope (Leica, TCS SP8). Captured images were processed and analyzed with ImageJ Software. At least 100 non-S-phase cells were randomly selected per sample. Data points presented in the text are the averages of intensities. Immunofluorescence Primary rat skin fibroblasts were fixed with 4% (vol/vol) paraformaldehyde in PBS for 15 min at room temperature, and then were permeabilized in 1 X PBS containing 0.1% Triton X-100 and blocked with blocking solution [1 X PBS containing 0.01% Triton X-100, 10% (vol/vol) FBS, and 3% (wt/vol) BSA] for 1 h. Primary antibodies in blocking solution were then added and incubated for 1 h at room temperature, followed by washing and incubation with fluorophore-conjugated corresponding secondary antibodies. Coverslips were counterstained and mounted on slides using mounting medium with DAPI. Images were acquired on a laser scanning confocal microscope (Cal Zeiss, LSM700). Immunolabeling of CPD The rat skin fibroblast primary cells were irradiated with 16 J/m2 UV-C and recovered by 1 h and 24 h. Approximately 7 X 104 primary cells plated on coverslips were washed twice with PBS and then fixed with 3% paraformaldehyde in PBS for 15 min. The cells were then permeabilized using 0.5% Triton X-100 in PBS for 10 min. For CPD immunolabeling, DNA was denatured using 0.1 M HCl in PBS for 15 min. Thereafter, the cells were incubated with PBS containing 0.5% BSA for 10 min. Immunolabeling of CPDs was performed using a mouse monoclonal antibody at a dilution of 1:1000. Western blotting For whole cell extracts, cells pellets were lysed in RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate] and Protease Inhibitor Cocktail (made in-house). 50 mg protein/lane was separated on 10%–12% Tris-Acetate gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, 10600002). Membranes were blocked in 5% skimmed milk in PBS-T [PBS, 0.1% (vol/vol) Tween20] for 1 h at room temperature and incubated with primary antibody [in 5% (wet/vol) skimmed milk in PBS-T] overnight at 4 C. Primary antibodies were listed in Key Resources Table. Antibody against alpha-tubulin served as loading controls. Membranes were washed three times in PBS-T, incubated with HRP-conjugated secondary antibody (Key Resources Table) in 5% (wt/vol) skimmed milk in PBS-T and visualized using MilliporeSigma Immobilon Western Chemiluminescent HRP Substrate (ThermoFisher Scientific, WBKLS0050). Immunohistochemistry Histological analysis of the cerebellum and cerebrum of CSBR571X/R571X rats was performed by using heterozygous CSBR571X/+ littermates as control. Rats were anesthetized with aether, and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. Brains were removed and sectioned into 3 mm slices before transfer into formalin. Tissues were fixed in 10% formalin for a minimum of 48 h at room temperature and then subjected to paraffin embedding schedule as follow: 70% Ethanol, two changes, 1 h each; 80% Ethanol, one change, 1 h; 95% Ethanol, one change, 1 h; 100% Ethanol, three changes, 1.5 h each; Xylene, three changes, 1.5 h each; Paraffin wax (58 C-60 C), two changes, 2 h each. After the paraffin wax cooled down and solidified, the paraffin blocks were trimmed and cut at 5 mm and placed in water bath at about 40 C-45 C. Sections were mounted into slides and air-dried for 30 minutes, then baked in 45 C oven overnight. Before deparaffinization, slides were baked in 65 C oven for 2 h. Slides were placed in a rack and subjected to deparaffinization and dehydration in the following washes: Xylene, two changes, 3 min each; Xylene 1:1 with 100% ethanol, one change, 3 min; 100% ethanol, two changes, 3 min each; 95% ethanol, one change, 3 min; 70% ethanol,

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one change, 3 min; 50% ethanol, one change, 3 min. Slides were kept in the tap water until ready to perform antigen retrieval. Slides were placed to a boil in antigen retrieval buffer [10 mM Sodium Citrate, 0.05% Tween20, pH 6.0], then maintained at a sub-boiling temperature for 10 min. Slides were cooled down in running tap water for 5 min. For H&E staining, slides were stained in hematoxylin for 3-5 min before antigen retrieval, and then washed in running tap water until sections blue for 5 min or less. Slides were differentiated in 1% acid alcohol (1% HCl in 70% alcohol) for 5 min and then washed in running tap water until the sections were again blue by dipping in an alkaline solution (ammonia water) followed by tap water wash. Slides were stained in 1% Eosin for 10 min and washed in tap water for 1-5 min. Slides were dehydrated through 95% alcohol, 2 changes of absolute alcohol, 5 min each, and cleared in 2 changes of Xylene, 5 min each. Finally, slides were mounted in mounting media. For cell-specific immunohistochemical staining, slides were blocked in blocking solution [1 X PBS-T containing 10% FBS, 1% BSA and 0.3% Triton X-100] for 1 h at room temperature after antigen retrieval. Slides were incubated in primary antibodies [diluted in 1 X PBS-T containing 1% BSA] overnight at 4 C. Slides were washed three times in PBS-T and incubated in fluorophore-conjugated secondary antibody diluted in PBS-T, 1% BSA for 1 h at room temperature. Slides were washed three times in PBS-T and mounted with mounting medium with DAPI. RNA sequencing Total RNA from 100 mg of fresh rat cerebellar cortex was isolated using the QIAGEN RNeasy Lipid Tissue Mini Kit. RNA concentration and integrity were assessed by a Nanodrop spectrophotometer (ThermoFisher Scientific) and an Agilent Bioanalyzer nanoChIP. RNA libraries were prepared by using the TruSeq Stranded Total RNA sample Preparation Kits (Illumina, San Diego, CA) following the manufacturer’s instructions. Purified libraries were quantified by a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Samples were sequenced on an Illumina HiSeq 2500 platform (San Diego, CA). All sequencing was performed at Novogene Lnc. QUANTIFICATION AND STATISTICAL ANALYSIS DEG analysis FastQC was conducted for Quality control (QC) of RNA-seq reads. Trimming was performed by seqtk for known Illumina TruSeq adaptor sequences, poor reads, and ribosomal RNA reads. The trimmed reads were then mapped to the Rattus norvegicus reference genome by the Hisat 2 (version: 2.0.4). String Tie (version: 1.3.0) was performed for each gene count from trimmed reads. Gene counts were normalized by trimmed mean of M-values, and fragments per kilobase of transcript per million mapped reads (FPKM) in Perl script. edgeR in R package was performed for determining differentially expressed genes and threshold with padj < 0.05 and absolute values of log2(Fold Change) > 1. Functional enrichment analysis GO pathways were enriched by R package (v 3.5.1) to better understand the functions of the DEGs. For all other experiments, data were collected in Microsoft Excel and statistical tests were performed in GraphPad Prism 6 software. Data were analyzed using two-tailed unpaired Student’s t test. For all analyses, P-values lower than 0.05 were considered statistically significant. Illustrations of these statistical analyses are displayed as the means ± standard deviation (SD). The exact values of ‘‘N’’ used are described in the corresponding figure legends. Unless otherwise stated in the figure legend, N refers to number of cells or biological replicates. DATA AND CODE AVAILABILITY The whole genome sequencing datasets generated in this study are available at Sequence Read Archive (SRA) https://www.ncbi. nlm.nih.gov/sra/?term= (Accession number: PRJNA550214). The RNA-seq datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) database https://www.ncbi.nlm.nih.gov/gds (Accession number: GSE126032).

e5 Cell Reports 29, 800–809.e1–e5, October 22, 2019