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An AUTS2–Polycomb complex activates gene expression in the CNS

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Abstract

Naturally occurring variations of Polycomb repressive complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, autism susceptibility candidate 2 (AUTS2). Although AUTS2 is often disrupted in patients with neuronal disorders, the mechanism underlying the pathogenesis is unclear. We investigated the role of AUTS2 as part of a previously identified PRC1 complex (PRC1–AUTS2), and in the context of neurodevelopment. In contrast to the canonical role of PRC1 in gene repression, PRC1–AUTS2 activates transcription. Biochemical studies demonstrate that the CK2 component of PRC1–AUTS2 neutralizes PRC1 repressive activity, whereas AUTS2-mediated recruitment of P300 leads to gene activation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) demonstrated that AUTS2 regulates neuronal gene expression through promoter association. Conditional targeting of Auts2 in the mouse central nervous system (CNS) leads to various developmental defects. These findings reveal a natural means of subverting PRC1 activity, linking key epigenetic modulators with neuronal functions and diseases.

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Figure 1: Characterization of the PRC1.5–AUTS2 complex.
Figure 2: Effect of AUTS2 on chromatin architecture and transcription.
Figure 3: CK2 inhibits H2A monoubiquitination activity of PRC1.5–AUTS2.
Figure 4: AUTS2 recruits P300 for gene activation.
Figure 5: Regulation of neuronal gene expression by AUTS2.
Figure 6: Effect of the Auts2 knockout on phenotypes associated with neurodevelopmental disorders.

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Gene Expression Omnibus

Data deposits

All ChIP-seq and RNA-seq data has been deposited into GEO database with the accession number GSE60411.

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Acknowledgements

We thank S. Zhang, R. Bonasio, S. Shen, and L. Cohen for assistance in analysis with genome-wide studies; H. Zheng for mass spectrometry analysis; L. Chiriboga, C. Loomis and the Experimental Pathology Cores at NYU Langone Medical Center for assistance in immunohistochemical analysis; D. Hernandez for assistance in various experiments. We appreciate the help of C. Guo and the transgenic facility at Howard Hughes Medical Institute’s (HHMI) Janelia Farm Research Campus in generating the Auts2 cKO mice. We are grateful to V. Bardwell and L. Di Croce for providing us PCGF1 and RING1B antibodies, respectively. We also thank A. Tarakhovsky for his generous help to our mouse work after Hurricane Sandy and L. Vales for valuable discussions and extensive reading of the manuscript. This work was supported as follows: the biochemical analysis of AUTS2 by the National Institute of Health grant GM-64844, and the mouse work by a Pilot Award from the Simons Foundation Autism Research Initiative (SFARI). A.S. is supported by grants from NIH (1DP2MH100012-01), Seaver Autism Foundation, and The Brain and Behavioral Research Fund Young Investigator Award (number 18194). Z.G. was supported by the SFARI pilot award and by an NIH training grant (5T32CA160002). P.L. was supported by an NIH postdoctoral fellowship (1F32GM105275). J.M.S was supported by an NIH postdoctoral fellowship (F32AA022842) as well as by the Simons Foundation as a Junior Fellow.

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Authors and Affiliations

Authors

Contributions

Z.G., P.L., J.M.S. and M.v.S. performed experiments. All authors contributed to data analysis, experimental design and manuscript writing.

Corresponding author

Correspondence to Danny Reinberg.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Requirement of the integrity of the PRC1–AUTS2 complex for transcriptional activation.

a, Luciferase activity in screened stable cell clones expressing GAL4–PCGF5, 24 h after induction by doxycycline at 100 μg ml−1. b, Fold change in luciferase activity in GAL4–AUTS2 cells upon knockdown of RING1B or PCGF5. Cells were transfected with Lipofectamine 2000 RNAiMAX and siRNAs against RING1B or PCGF5, or control siRNAs for two days and then 100 μg ml−1 doxycycline was added to induce GAL4–AUTS2 expression. Then 24 h after induction, luciferase activity was measured. Each value is the mean of three independent measurements. Error bars represent standard error. c, Immunoblotting of samples used for luciferase activity reporter assay as in b using the antibodies indicated. d, Fold change in luciferase activity in GAL4–PCGF5 cells upon knockdown of AUTS2. Cells were treated as in b. e, Immunoblotting of samples used for luciferase activity reporter assay as in d using the antibodies indicated.

Extended Data Figure 2 H2A monoubiquitination assay and CK2 kinase assay performed with [γ-32P]ATP.

a, Coomassie blue staining of factors used. b, Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (see Methods for details). c, Immunoblotting of H2A monoubiquitination assay as described in b with increasing amounts of CK2. d, Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37 °C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS–PAGE. Besides CK2B, which was radio-labelled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e, H2A monoubquitination assay performed as in Fig. 3c, using increasing amounts of RING1B–PCGF5–AUTS2 containing either RING1B(S41A) (S41 to alanine), or RING1B(S41D) (S41 to aspartic acid), purified from Sf9 cells.

Extended Data Figure 3 Interaction of AUTS2 and P300.

a, Immunoprecipitation from nuclear extract of 293T cells expressing NFH–AUTS2 using AUTS2 antibody, followed by western blotting for the antigens indicated. b, Immunoprecipitation using recombinant proteins of P300 and AUTS2 purified from Sf9 cells and a P300 antibody, followed by western blotting using antibodies against P300 and AUTS2.

Extended Data Figure 4 Luciferase activity without normalization.

a, b, Analysis using data from Fig. 4b, c, respectively.

Extended Data Figure 5 Expression of AUTS2 in mouse brain.

a, Validation of AUTS2 antibody by immunohistochemistry (IHC) in NFH-AUTS2 stable cells. Upon doxycycline induction, a stronger nuclear staining was detected compared with non-induction control, confirming the antibody we raised is suitable for IHC. b, Detection of AUTS2 protein in a mouse embryo at E15 by IHC with AUTS2 antibody. c, IHC analysis of a sagittal brain section from an adult mouse using AUTS2 antibody. d, Expression of AUTS2 in the mouse brain. Immunoblotting was performed with whole brain extracts at various developmental stages as indicated. e, Immunofluorescence staining of AUTS2 in P3 mouse brain. AUTS2 expression is confined to neurons (top panels) as seen by co-localization with the neuronal marker NeuN in the cortex and hippocampus. AUTS2 does not co-localize with the glial marker GFAP (bottom panels) in the same regions.

Extended Data Figure 6 Genome-wide analysis of AUTS2 ChIP-seq signals.

a, HOMER was used to compute the genomic distribution of AUTS2 peaks obtained from AUTS2 ChIP-seq in mouse brain. b, Histogram of the distribution of AUTS2 peaks relative to TSS, calculated via HOMER. c, Percentage of AUTS2 target genes overlapped with highest (top 25%, red bar) and lowest (bottom 25%, green bar) expression levels of all genes in mouse brain. d, Percentage of overlapped peaks between two biological replicates of AUTS2 ChIP-seq in mouse brain.

Extended Data Figure 7 P300 is localized to AUTS2 targeted loci in mouse brain.

a, IGV browser views for input, P300, AUTS2, Pol II, H3K27ac, H3K4me3, H3K36me3, H3K27me3, and H2AK119ub1 ChIP-seq performed in P1 mouse brain at two representative loci. The y axis corresponds to ChIP-seq signal intensity. Gene representation at each locus is shown at the bottom. b, ChIP reads density plots for levels of P300 at loci co-targeted by AUTS2/RING1B and BMI1/RING1B. A ± 1 kb window relative to the centre of peaks is shown.

Extended Data Figure 8 ChIP-seq in 293 T-REx cells.

a, IGV browser views for input, HA–AUTS2, HA–RING1B, Pol II, H3K27ac, H3K4me3, H3K36me3, HA–CBX2, and H3K27me3 ChIP-seq libraries at two representative loci. The y axis corresponds to the ChIP-seq signal intensity. Gene representation at each locus is shown at the bottom. ChIP-seq data for HA–RING1B, HA–CBX2, and H3K27me3 obtained from a previous study16. b, Genomic distribution of HA–AUTS2 target regions relative to TSS. The x axis corresponds to the distance from TSS (−20 kb to +20 kb); the y axis corresponds to frequency. ce, Venn diagrams showing the overlap among regions targeted by factors as indicated. f, Analysis of mRNA levels of top targets identified by HA–AUTS2 ChIP-seq in 293 T-REx cells. RT–qPCR using the primers indicated was performed from vector control (mock) or NFH–AUTS2 stable cell lines induced by doxycycline (+NFH–AUTS2). All values are the mean of three technical replicates and error bars represent standard deviation.

Extended Data Figure 9 Generation of mice with Auts2 conditional knockout in the nervous system and additional developmental phenotypes.

a, ES cells carrying an engineered allele of Auts2 were generated through homologous recombination. Specifically, two LoxP sites were placed flanking exon 7 of Auts2. A cassette containing SA–IRES–tdTomato and an inverted PGK–Neo (neomycin phosphotransferase gene) were flanked by two FRT (FLP recombinase target) sites and inserted between the first LoxP site and exon 7. A WPRE (woodchuck hepatitis post-transcriptional regulatory element) sequence was placed immediately downstream of tdTomato to enhance its expression. Homologous mice carrying this engineered sequence are expected to give rise to a transcript containing only the first six exons of Auts2 followed by IRES-driven tdTomato. Red fluorescence serves as a marker for successful gene targeting. To obtain the conditional deletion of Auts2, these mice were crossed with FLP mice to excise the FRT flanking sequence, resulting in floxed mice, which were then crossed with nestin-Cre deleter mice to generate Auts2 deletion in the nervous system. b, Genotyping of the Auts2 flox mice by PCR. The fast migrating species of 225 bp represents the PCR product of wild type, and the species of 317 bp corresponds to the knockout. c, Knockout mice are significantly shorter than both heterozygous and wild-type mice across development. #Post-hoc difference (P < 0.05) between wild type and knockout. d, The KO mice took significantly longer to orient their nose to an upward position. e, No significant difference in body weight was detected at P1, however, a significantly reduced milkband was observed in Auts2 knockout.

Extended Data Figure 10 Altered expression of genes targeted by PRC1–AUTS2 in brains of Auts2 knockout mice.

a, Expression profiles of select genes simultaneously targeted by AUTS2 and RING1B (labelled as AUTS2+ RING1B+). As negative control, two non-target genes were used (labelled as AUTS2− RING1B−). Total RNAs were extracted from whole brains of either wild-type or knockout mice, followed by reverse transcription to generate cDNAs for RT–qPCR. Expression levels are normalized over those in wild type. All mean values of expression levels and standard errors were calculated from duplicated measurements of three biological replicates. *P < 0.05 by two-sided t-test. b, IGV views of four representative loci for genes examined as in a, showing the enrichment of AUTS2, RING1B, Pol II, and active histone marks.

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Gao, Z., Lee, P., Stafford, J. et al. An AUTS2–Polycomb complex activates gene expression in the CNS. Nature 516, 349–354 (2014). https://doi.org/10.1038/nature13921

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