Single-cell transcriptomic profiling of mouse KrasG12DP53-/- (KP) lung tumors in response to tumor cell intrinsic Asf1a knockout (KO), anti-PD-1 or combination treatment (Asf1a KO + anti-PD-1).
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ABSTRACT: We performed single-cell RNA sequencing (scRNAseq) analysis on the mouse lung tissues from KP tumor-bearing mice treated with tumor cell intrinsic Asf1a KO, anti-PD-1 or combination treatment
Project description:KrasG12D/P53-/-(KP)-Cas9 single clone was transfected with ctrl vector, ctrl sgRNA-1, ctrl-sgRNA-2 or ctrl-sgRNA-3 and then selected by using 5ug/ml Blasticidin, to get the 4 ctrl lines; KP-Cas9 single clone was transfected with Asf1a sgRNA-1, sgRNA-2, sgRNA-3 and new sgRNA-1 and then selected by using 5ug/ml Blasticidin, and then picked up single clones with Asf1a KO. For each Asf1a sgRNA, we selected one clone for RNA seq, so totally we have 4 Asf1a KO clones for RNA seq.
Project description:Despite substantial progress in lung cancer immunotherapy, the overall response rate in KRAS-mutant lung adenocarcinoma (ADC) patients remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses—such as epigenetic modulation of anti-tumor immunity—is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused sgRNA library, and performed an in vivo CRISPR screen in KrasG12D/P53-/- (KP) lung ADC model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of Asf1a inhibition and anti-PD-1 immunotherapy.
Project description:To investigate the chemokines expressed by the KP cancer line and to compare it to chemokines expressed in lung tissues upon inoculation of KP tumor cells
Project description:YAP depletion in the KP tumor system results in smaller tumors and delayed tumor latency. We used microarrays to investigate changes in global gene expression due to YAP1 loss in KP tumors
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites.
Project description:Bivalent chromatin domains containing both repressive H3K27me3 and active H3K4me3 modifications are barriers for the expression of lineage-specific genes in embryonic stem (ES) cells and must be resolved for the transcription induction of these genes during differentiation, a process that remains largely unknown. Here, we show that Asf1a, a histone chaperone involved in both nucleosome assembly and disassembly, regulates the resolution of bivalent domains and activation of lineage-specific genes during mouse ES cell differentiation. Deletion of Asf1a does not affect the silencing of pluripotent genes, but compromises the expression of lineage-specific genes during ES cell differentiation. Mechanistically, the Asf1a-histone interaction, but not the role of Asf1a in nucleosome assembly is required for gene transcription. Asf1a is recruited to several bivalent promoters, partially through association with transcription factors, and mediates nucleosome disassembly during differentiation. We suggest that Asf1a-mediated nucleosome disassembly provides a means for resolution of bivalent domain barriers for induction of lineage-specific genes during differentiation.