Project description:ChIP-Seq of C11orf53(OCA-T), COLCA2 and POU2F3(OCT11) in tuft cell lung cancer cell lines. Gene expression changes upon CRISPR knockout of different genes in tuft cell lung cancer cell lines and upon over-expression of C11orf53/POU2F3 alone or in combo in YT330 (a murine Notch high small cell lung cancer cell line)
Project description:Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), functions as a new co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrated that POU2AF2 functions as an “Yin-Yang” factor which mediates both transcriptional activation and repression at distal enhance elements. The proteomics study aims to identify potential interactors with POU2AF2 in different cell lines which may enable us to further understand the function of POU2AF2 in human cancer.
Project description:Small cell lung cancer (SCLC), accounting for around 15% of all lung cancers, often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. The POU class 2 homeobox 3 (POU2F3) is a master regulator of tuft cell identity and defines a subtype of SCLC tumors (SCLC-P subtype) that lack the expression of neuroendocrine markers. Here, we have identified a previously uncharacterized protein C11orf53, which is co-expressed with POU2F3 in both SCLC cell lines and patient samples, and defines the same SCLC subtype. C11orf53 is an essential gene for SCLC-P subtype cells’ survival based on genome wide CRISPR screen, and genetic depletion of C11orf53 or POU2F3 induces apoptosis in different SCLC-P subtype cell lines. Furthermore, our biochemical and genome-wide studies have demonstrated that C11orf53 is a novel chromatin-bound protein that occupies broad active enhancer domains and regulates gene expression at active enhancers. Mechanistically, C11orf53 directly interacts with POU2F3 at the POUs domain, and is recruited to chromatin by POU2F3. Depletion of C11orf53 dramatically reduced enhancer H3K27ac levels and chromatin accessibility, resulting a reduction of the expression of POU2F3-dependent tuft cell markers. Based on the expression pattern and molecular function, we have renamed C11orf53 as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2). In summary, our study has identified a new co-activator of POU2 family transcription factors, and sheds light on the therapeutic potential of targeting POU2AF2/POU2F3 heterodimer in human SCLC.
Project description:~12% of SCLCs are marked by the lineage transcription factor POU2F3, which is essential in all POU2F3-positive SCLCs. Thus, approaches to directly or indirectly inhibit POU2F3 could lead to new therapeutic strategies for POU2F3-positive SCLCs. Here we use a positive selection screening strategy where endogenous POU2F3 is fused to the suicide gene DCK*. Cells that express endogenous POU2F3-DCK* are killed in the presence of the nucleoside analog BVdU and only cells that downregulate the POU2F3-DCK* fusion survive. Genome-wide CRISPR/Cas9 resistance screens with BVdU in POU2F3-DCK* cells uncovered that inactivation of SMARCD1 or BRD9, both components of the non-canonical BAF (ncBAF) complex, markedly downregulate endogenous POU2F3. We find that all POU2F3-positive SCLC cell lines relative to ASCL1-positive and NEUROD1-positive cell lines, are exquisitely sensitive to mSWI/SNF complex inhibition using SMARCA2/4 inhibitors; while pure non-neuroendocrine POU2F3-positive SCLC cell lines are highly sensitive to ncBAF complex inhibition using highly selective BRD9 degraders. Mechanistically, BRD9 binds and regulates POU2F3 target genes including POU2F3 itself. BRD9 degraders or SMARCA2/4 inhibitors robustly decrease accessibility of POU2F3 target genes effectively shutting off POU2F3 function. Moreover, BRD9 degraders or SMARCA2/4 inhibitors decrease tumor growth and increase survival of mice bearing non-neuroendocrine POU2F3 xenografts. This works shows that mSWI/SNF and ncBAF tightly regulate POU2F3 expression and activity and nominate mSWI/SNF or ncBAF inhibition as druggable therapeutic strategies to selectively target POU2F3-positive SCLCs.
Project description:OCA-B, OCA-T1, and OCA-T2 belong to a family of transcriptional coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IkBz (encoded by the NFKBIZ gene) as the fourth member of the OCA protein family. While originally discovered as an inducible regulator of NFkB, we show here that IkBz shares a microhomology with OCA proteins and uses this segment to simultaneously bind to POU transcription factors and octamer motif-containing DNA. Our functional reporter assays suggest that IkBz requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by our epigenomic analysis of MYD88 L265P-mutant lymphoma cells, which revealed colocalization of IkBz, the POU transcription factor OCT2, and NFkB:p50 at hundreds of DNA elements harboring octamer and kB motifs. These results suggest that IkBz is a transcriptional coactivator that integrates and amplifies the output of NFkB and POU transcription factors at inducible genes in immune cells.
Project description:Total gallbladder/extrahepatic duct tissue digest from Pou2f3-/- (KO) and Pou2f3+/+, Pou2f3+/- (heterozygous and homozygous WT mice together in WT sample) littermated mice were sorted for live cells on Moflo XDP cell sorter (singlets, FSCAxSSCA, DAPI-). Immediately post-sorting, cells were run on 10X Chromium (10X Genomics) and then through library preparation by the Institute for Human Genetics at UCSF following the recommended protocol for the Chromium Single Cell 3′ Reagent Kit (v3 Chemistry). Libraries were run on the NovaSeq 6000 for Illumina sequencing. Post-processing and quality control were performed by the Genomics Core Facility at the Institute for Human Genetics at UCSF using the 10X Cell Ranger package (Cell Ranger Version 3.0.2, 10X Genomics). Primary assessment with this software for WT DC sample reported 2,441 cell-barcodes with 7,651 median unique molecular identifiers (UMIs, transcripts) per cell and 2,004 median genes per cell sequenced to 61.4% sequencing saturation with 48,559 mean reads per cell. Primary assessment with this software for MARCH1-/- DC sample reported 681 cell-barcodes with 6,309 median unique transcripts per cell and 1,774 median genes per cell sequenced to 73.1% sequencing saturation with 202,410 mean reads per cell.