Work Description

Date: 4 October, 2024 Dataset Title: Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis Dataset Creators: Masayoshi Nagai, Robert S. Porter, Maxwell Miyasato, Aijia Wang, Cecilia M. Gavilan, Elizabeth D. Hughes, Michael C. Wu, Thomas L. Saunders, and Shigeki Iwase Dataset Contact: Shigeki Iwase siwase@umich.edu Funding: This work was supported by National Institutes of Health (NIH) grants (R01NS116008, R21NS12544, R21MH127485 to S.I.) and the University of Michigan?Pandemic Research Recovery Program (U078150, to M.N.). The work in the Michigan Transgenic Core was supported by the National Cancer Institute of the National Institutes of Health under award number P30CA046592. Key Points: � The asynchronous splicing of LSD1 and PHF21A resulted in the formation of an intermediate complex consisting of neuronal PHF21A (PHF21A-n) and canonical LSD1 (LSD1-c) prior to the mature neuronal complex consisting of PHF21A-n and LSD1-n. � The H3K4 demethylation activity of the complex diminishes in a stepwise manner during this maturation process. The enzymatic activity of the mature complex was undetectable. � Multiplex proteomics study revealed that, though enzymatically inactive, PHF21A and LSD1 neuronal isoforms interact with unique proteins in neurons compared to non-neuronal cells. � These neuron-specific interaction partners include MYT1-family transcription factor and, unexpectedly, post-transcriptional mRNA processing factors such as VIRMA and CPSF6. � With a new mouse model in which PHF21A-c is expressed in neurons, we further tested whether PHF21A neuronal exon is required for its interaction with the novel neuron-specific binding partners and found that the PHF21A neuronal exon is dispensable. Thus, PHF21A and LSD1 form neuron-specific complexes by virtue of the neuronal proteomic milieu rather than the neuronspecific protein segments generated by the alternative splicing. Research Overview: PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis. Methodology: This dataset contains raw data and numerical values for all graphs in "Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis". Fig.1C, 1F, 1G, SFig.2A,B and 2D are quantitative data of Western Blotting results. Fig.4B, 4C, 5H, 5I, 5K and SFig.3C are results of IP-MS. Fig.6B, 6C, 6E and 6F are results of synapses. Fig. 6H, 6I, 6K and 6L are results of dendrites. SFig.1B and 1C,D are results of RT-pPCR. SFig. 1E, 1F and 1G are results of Complete Amplicon Seqence. Files contained here: All files are used to create graphs and quantify data. The files and simulations are described below: Data-for-Fig 2024-10-03_All low data including charts. Fig.1C_ Expression of PHF21A and associated proteins in LUHMES cells and 293T cells examined by Western blot analysis using antibodies as indicated. Fig.1F_ Quantification of Western signals for PHF21A-c, PHF21A-n, and LSD1 normalized by histone H3. Fig.1G_ The ratio of PHF21A protein isoforms in the developing mouse brain based on the Western signals. Fig.4B_ Co-IP-MS analysis of PHF21A-associated proteins in MEF. Fig.4C_ Co-IP-MS analysis of PHF21A-associated proteins in cortical neurons. Fig.5H_ Volcano plots of Co-IP-MS analysis of PHF21A-interacting proteins using Phf21a+/+ cortices. Fig.5I_ Volcano plots of Co-IP-MS analysis of PHF21A-interacting proteins using Phf21a��n/��n cortices. Fig.5K_ Scatter plot comparing the log2FC (a-PHF21A antibody/control IgG) of all PHF21A-associated proteins between WT and Phf21a��n/��n cortices. Fig.6B_ Excitatory synapses density in cultured cortical neurons in Phf21a-null mutants. Fig.6C_ Excitatory synapses density in cultured cortical neurons in Phf21a-��n mutants. Fig.6E_ Inhibitory synapses density in cultured cortical neurons in Phf21a-null mutants. Fig.6F_ Inhibitory synapses density in cultured cortical neurons in Phf21a-��n mutants. Fig.6H_ Quantification of dendritic length in Phf21a-null mutants. Fig.6I_ Quantification of dendritic length in Phf21a-��n mutants. Fig.6K_ Quantification of dendritic spine density in Phf21a-null mutants. Fig.6L_ Quantification of dendritic spine density in Phf21a-��n mutants. SFig.1B_ RT-pPCR analyses for the neuronal markers RBFOX3 (NEUN) and TUBB3 validate the neuronal differentiation of LUHMES cells. SFig.1C,D_ C; PCR efficiency of LSD1 mRNA isoforms empirically determined by the LSD1-c and LSD1-n cDNA-carrying plasmids. D; Correction values of LSD1 isoform expression after 30 cycles. SFig.1E_ The ratio of LSD1-c and LSD1-n mRNA isoforms. Cells were differentiated into neurons as indicated and harvested on day 3 to day 12. SFig.1F_ The ratio of LSD1-c and LSD1-n mRNA isoforms in the developing mouse brain. SFig.1G_ The ratio of LSD1-c and LSD1-n mRNA isoforms in cultured MEF or cortical neurons. SFig.2A,B_ A; The WTN signals of H3K4me1. B; The WTN signals of H3K4me2. SFig.2D_ Cellular fractionation assays were used to evaluate the chromatin binding of the PHF21A-LSD1 complex at day 0 and day 3 of LUHMES cell differentiation. SFig.3C_ Volcano plot comparing the proteins immunoprecipitated by PHF21A antibody from Phf21a+/+ vs. Phf21a��n/��n brains. Related publication(s): Nagai, M., Porter, R. S., Miyasato, M., Wang, A., Gavilan, C. M., Hughes, E. D., Wu, M. C., Saunders, T. L., Iwase, S. Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis. Journal of Biological Chemistry (2024) Use and Access: This data set is made available under a Creative Commons Public Domain license (CC0 1.0). To Cite Data: Nagai, M., Porter, R. S., Miyasato, M., Wang, A., Gavilan, C. M., Hughes, E. D., Wu, M. C., Saunders, T. L., Iwase, S. Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis [Data set]. University of Michigan - Deep Blue. https://doi.org/10.7302/y4bw-4n85