Effect of ASCL1 phosphorylation status on chromatin binding and target regulation in neuroblastoma
ASCL1, a basic helix-loop-helix family transcription factor, is a master regulator of developmental neurogenesis and a crucial component of transcription factor cocktails that can convert heterologous cell types such as fibroblasts into neurons. The ability of ASCL1 to drive neuronal differentiation is controlled by multi-site phosphorylation but how this modification controls the genome-wide transcriptional activity of ASCL1 is unknown.  Using human neuroblastoma cells that maintain a rapidly dividing neuroblastic phenotype yet retain the ability to undergo differentiation as a model system, we find that phosphorylation of ASCL1 has limited effect on target gene promoter association, but predominantly regulates  its binding to a subset of distal enhancer regions, resulting in extensive differences in target control by activation, as well as direct and indirect gene repression.  These genome-wide analyses reveal how post-translational modification of ASCL1 can change its structure and function, driving it to differential regulatory elements to change cell fate, and controlling ASCL1's activity as a master transcription regulator of neurogenesis. Using functional mutational and pharmacological approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells results in co-ordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating a gene programme driving neuronal differentiation. Thus, we show targeting the post-translational modification of a key developmental regulator can re-engage  a latent genome-wide programme forcing mitotic exit and differentiation in cancer cells. Overall design: RNA-seq analysis of SH-SY5Y ASCL1-doxycycline-inducible cells. 5 replicates each of 2 unique clones were used, for uninduced, WT-induced, and S-A induced cells. ASCL1 ChIP-seq analysis of SH-SY5Y ASCL1-doxycycline-inducible cells. 3 replicates each of 2 unique clones were used , for WT-induced, and S-A induced cells, plus input for each sample. Generation of lentivirally transduced cell lines: Viral constructs were generated by site directed mutagenesis using the QuikChange II XL Site- Directed Mutagenesis Kit (Agilent Technologies) and cloned into a 3rd Generation Lenti-X vector (pLVX-TREG) (Clontech) using In-Fusion® HD Cloning Kit (ClonTech). Viral constructs were made by transfecting Lenti-X™ 293T cell line (Clontech) with 250mM calcium phosphate and the vector of interest, and packaging mix: PMD2G, PMLg, REV/PRSV in a ratio of 6:3:4:2 respectively. Viruses were concentrated using LenitiX concentrator (Clontech) and titered using the Lenti-XTM qRT-PCR Titration Kit (ClonTech). Neuroblastoma cells were transduced with a Tet-On transactivator (pLVX-CMV-Tet3G) (Clontech) at an MOI of 10. After 48 hours, cells were selected with 500 µg/ml G418 for 72 hours based on a previously determined optimal antibiotic kill curve. These cells were then transduced with pLVX-TREG - encoding either human WT or S-A ASCL1 at a multiplicity of infection of 10 and then selected after 48 hours with 1 µg/ml puromycin for 72 hours based on previously determined kill curves. ChIP-seq: ChIPseq was performed as described previously (Jozwik et al., 2016; Schmidt et al., 2009) using anti-ASCL1 (Abcam, cat# ab74065).  ChIPseq experiments were performed in two separate clones of each of WT and S-A SH-SY5Y tetracycline-inducible stable cell lines induced with 1 µg/ml doxycycline for 24 hours, before cross-linking with 1% formaldehyde. ChIPseq experiments were performed in at least four biological replicates. ChIP-seq and the input libraries were prepared using the ThruPLEX® DNA-seq Kit (Rubicon Genomics). Reads were mapped to hg19 genome using Bowtie2 2.2.6. Aligned reads with the mapping quality less than five were filtered out. The read alignments from four replicates were combined into a single library, and peaks were called with model based analysis for ChIP-seq 2 (MACS2) version 2.0.10.20131216 using sequences from SH-SY5Y chromatin extracts as a background input control. Meme version 4.9.1 was used to detect known and discover binding motifs among tag-enriched sequences. For visualising tag density and signal distribution heatmap, the normalized read coverage in a window of a ±2.5- or 5-kb region flanking the tag midpoint was generated using a bin size of 1/100 of the window length. Differential binding analysis (Diffbind) was performed as described previously (Jozwik et al., 2016). RNA-seq: RNA-sequencing experiments were performed in SH-SY5Y, WT and S-A SH-SY5Y tetracycline-inducible stable cell lines, using two clones of each. For the inducible cell lines, cells were induced with 1 µg/ml doxycycline for 24 hours and RNA extracted. Experiment was performed in at least five biological replicates for each cell line. Single-end 40-bp reads generated on the Illumina HiSeq sequencer were aligned to the human genome version GRCh37 and read counts were generated using STAR 2.5.1a (Anders and Huber, 2010).