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Interferons (IFNs) are cytokines with potent antineoplastic and antiviral properties. IFNα has significant clinical activity in the treatment of myeloproliferative neoplasms (MPN), but the precise mechanisms by which it acts are not well understood. Here, we demonstrate that chromatin assembly factor 1 subunit B (CHAF1B), an Unc-51-like kinase 1 (ULK1)-interactive protein in the nuclear compartment of malignant cells, is overexpressed in patients with MPN. Remarkably, targeted silencing of CHAF1B enhances transcription of IFNα-stimulated genes and promotes IFNα-dependent antineoplastic responses in primary MPN progenitor cells. Taken together, our findings indicate that CHAF1B is a promising newly identified therapeutic target in MPN and that CHAF1B inhibition in combination with IFNα therapy might offer a novel strategy for treating patients with MPN. Significance: Our findings raise the potential for clinical development of drugs targeting CHAF1B to enhance IFN antitumor responses in the treatment of patients with MPN and should have important clinical translational implications for the treatment of MPN and possibly in other malignancies.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinasessup1-5/sup. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUTamp;Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

In the process of urbanisation in China, many cities expect to promote the development of new towns through the construction of sports centres. However, due to the unreasonable design of the scale, accessibility and vitality of the sports centre, the catalyst effect of the sports centre is difficult to play an effective role. Using cluster analysis algorithm and spatial syntax, this study analyses the algorithm of sports centres with sustainable development characteristics, and puts forward the conclusions of sustainable development planning strategies such as venue merger design under the principle of intensive, maintaining high accessibility of sports centres, ensuring the compound function of surrounding plots and grid space to improve regional vitality, in order to provide some inspiration for the planning and construction of Sports Centre in the future.

CHAF1B interacts with ULK1 in the nuclear compartment of JAK2V617F-positive cells and is overexpressed in patients with MPN. A and B, Left, ULK1–protein complexes were co-IP using an anti-ULK1 antibody from cytosolic and nuclear protein fractions isolated from untreated or IFNα-treated (10 or 240 minutes) HEL (A) or SET-2 (B) cells and then resolved by SDS-PAGE. As control, cytosolic and nuclear lysates isolated from cells treated with IFNα for 240 minutes were incubated with normal rabbit IgG (RIgG) antibody. Interaction between ULK1 and CHAF1B was assessed by immunoblotting with anti-ULK1 and anti-CHAF1B antibodies. Note: ǂ, unspecific band. A and B, Right, Equal amounts of cytosolic and nuclear protein lysates isolated from untreated and IFNα-treated HEL (A) and SET-2 (B) cells used for co-IPs were resolved by SDS-PAGE, transferred to PVDF membranes and then immunoblotted with antibodies against ULK1, CHAF1B, α-tubulin (cytosolic marker), and lamin A/C (nuclear marker), as indicated. A and B, Blots are representative of three independent experiments. C, Scatter dot plot of log2CHAF1B mRNA expression in neutrophils from healthy individuals (normal, n = 11) and patients with ET (n = 47), PMF (n = 18), and PV (n = 28). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Shown are means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett multiple comparisons test to assess P values between patients with MPN and healthy individuals. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Scatter dot plots of log2CHAF1B mRNA expression in neutrophils from patients with ET (green triangles), PMF (purple diamonds), and PV (red squares) carrying wild-type (WT) or mutant (MUT) JAK2 (D), CALR (E), and TET2 (F) genes (JAK2 WT: ET n = 20, PMF n = 10, PV n = 5; JAK2 MUT: ET n = 26, PMF n = 8, PV n = 23; CALR WT: ET n = 27, PMF n = 11, PV n = 26; CALR MUT: ET n = 15, PMF n = 5, PV n = 1; TET2 WT: ET n = 45, PMF n = 17, PV n = 24; TET2 MUT: ET n = 2, PMF n = 1, PV n = 4). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Patients for which no information was available for the mutational status for the JAK2, CALR, or TET2 genes were excluded from the analysis. Shown are means ± SEM. Statistical analyses were performed using two-sample two-tailed t test: *, P < 0.05.

Gene-targeted inhibition of CHAF1B increases IFNα-inducible mRNA expression of ISGs in JAK2V617F-positive leukemic cells. qRT-PCR analyses of the indicated genes in control (Ctrl) siRNA or CHAF1B siRNA-transfected JAK2V617F-positive HEL (A) and SET-2 (B) cells, either left untreated or treated with IFNα for 6 hours. GAPDH mRNA expression was used for normalization. Data are expressed as fold change over control siRNA-transfected untreated cells (control) and represent means ± SEM of three independent experiments for each cell line. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, ChIP assay was performed in untreated and IFNα-treated (for 6 hours at 5,000 IU/mL) HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Immunoblot analysis of ULK1 expression in control siRNA (Ctrlsi) and ULK1 siRNA (ULK1si) transfected HEL cells either left untreated or treated with IFNα (for 6 hours at 5,000 IU/mL), as indicated. GAPDH levels were used as loading control. Blots are representative of three independent experiments used to perform ChIP assays shown in E. E, ChIP assay was performed in untreated (UT) and IFNα-treated control (Ctrl) siRNA- and ULK1 siRNA-transfected HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. F, HEL cells were pretreated for 1 hour with either DMSO (Ctrl and IFNα groups) or SBI-0206965 (SBI; 10 μmol/L; SBI and SBI+IFNα groups) followed by 6 hours of treatment with either DMSO (Ctrl), SBI (10 μmol/L), IFNα (5,000 IU/mL) or SBI+IFNα, as indicated. ChIP assay was performed in HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. E and F, IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using two-way ANOVA followed by Tukey multiple comparisons test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Relevant statistical differences are shown for the binding of CHAF1B to ISG15 and IFIT1 promoter regions between experimental conditions.

Biological processes in which putative ULK1-protein complexes are involved in the cytosol.

Inhibition of CHAF1B increases IFNα-induced anticlonogenic effects in MPN. HEL cells (A) and SET-2 cells (B) were transfected with either control (Ctrl) siRNA or CHAF1B siRNA, and leukemic CFU-L colony formation was assessed in clonogenic assays in the presence or absence of IFNα, as indicated. Data are expressed as percentage of colony formation over control siRNA-transfected untreated cells (control), and the scatter dot plots represent means ± SEM of four independent experiments for each cell line. C, Clonogenic capability of primary malignant erythroid progenitors isolated from PV patients transfected with either control (Ctrl) siRNA or CHAF1B siRNA and either left untreated or treated with IFNα, as indicated. Data are expressed as percentage of colony formation over control siRNA-transfected untreated cells (control) and shown are means ± SEM of six independent experiments using cells from six different patients with PV. Percentages for the same patient are represented by the same symbol and different patients have different symbols. A–C, Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Interferons (IFNs) are cytokines with potent antineoplastic and antiviral properties. IFNα has significant clinical activity in the treatment of myeloproliferative neoplasms (MPN), but the precise mechanisms by which it acts are not well understood. Here, we demonstrate that chromatin assembly factor 1 subunit B (CHAF1B), an Unc-51-like kinase 1 (ULK1)-interactive protein in the nuclear compartment of malignant cells, is overexpressed in patients with MPN. Remarkably, targeted silencing of CHAF1B enhances transcription of IFNα-stimulated genes and promotes IFNα-dependent antineoplastic responses in primary MPN progenitor cells. Taken together, our findings indicate that CHAF1B is a promising newly identified therapeutic target in MPN and that CHAF1B inhibition in combination with IFNα therapy might offer a novel strategy for treating patients with MPN.Significance:Our findings raise the potential for clinical development of drugs targeting CHAF1B to enhance IFN antitumor responses in the treatment of patients with MPN and should have important clinical translational implications for the treatment of MPN and possibly in other malignancies.

Multi-isotope systems have shown great application potential in tracing geological and environmental processes. In order to obtain the isotopic composition of multiple elements of interest, the common protocol is to separate each element from the matrix by independent procedures, which has some limitations, including poor efficiency, being time-consuming, requiring large samples and being unsuitable for rare samples (

Recent efforts have shown that structural variations (SVs) can disrupt three-dimensional genome organization and induce enhancer hijacking, yet no computational tools exist to identify such events from chromatin interaction data. Here, we develop NeoLoopFinder, a computational framework to identify the chromatin interactions induced by SVs, including interchromosomal translocations, large deletions and inversions. Our framework can automatically resolve complex SVs, reconstruct local Hi-C maps surrounding the breakpoints, normalize copy number variation and allele effects and predict chromatin loops induced by SVs. We applied NeoLoopFinder in Hi-C data from 50 cancer cell lines and primary tumors and identified tens of recurrent genes associated with enhancer hijacking. To experimentally validate NeoLoopFinder, we deleted the hijacked enhancers in prostate adenocarcinoma cells using CRISPR–Cas9, which significantly reduced expression of the target oncogene. In summary, NeoLoopFinder enables identification of critical oncogenic regulatory elements that can potentially reveal therapeutic targets. This work presents NeoLoopFinder, a computational method, for identifying chromatin interactions of structurally rearranged genomes. NeoLoopFinder was applied in 50 cancer datasets and identified genes associated with enhancer-hijacking events.

Heart regeneration is negligible in humans and mammals but remarkable in some ectotherms. Humans and mammals lack nucleated red blood cells (NRBCs), while ectotherms have sufficient NRBCs. This study used Bufo gargarizan gargarizan, a Chinese toad subspecies, as a model animal to verify our hypothesis that NRBCs participate in myocardial regeneration. NRBC infiltration into myocardium was seen in the healthy toad hearts. Heart needle-injury was used as an enlarged model of physiological cardiomyocyte loss. It recovered quickly and scarlessly. NRBC infiltration increased during the recovery. Transwell assay was done to in vitro explore effects of myocardial injury on NRBCs. In the transwell system, NRBCs could infiltrate into cardiac pieces and could transdifferentiate toward cardiomyocytes. Heart apex cautery caused approximately 5% of the ventricle to be injured to varying degrees. In the mildly to moderately injured regions, NRBC infiltration increased and myocardial regeneration started soon after the inflammatory response; the severely damaged region underwent inflammation, scarring, and vascularity before NRBC infiltration and myocardial regeneration, and recovered scarlessly in four months. NRBCs were seen in the newly formed myocardium. Enzyme-linked immunosorbent assay and Western blotting showed that the levels of tumor necrosis factor-α, interleukin- 1β, 6, and11, cardiotrophin-1, vascular endothelial growth factor, erythropoietin, matrix metalloproteinase- 2 and 9 in the serum and/or cardiac tissues fluctuated in different patterns during the cardiac injury-regeneration. Cardiotrophin-1 could induce toad NRBC transdifferentiation toward cardiomyocytes in vitro. Taken together, the results suggest that the NRBC is a cell source for cardiomyocyte renewal/regeneration in the toad; cardiomyocyte loss triggers a series of biological processes, facilitating NRBC infiltration and transition to cardiomyocytes. This finding may guide a new direction for improving human myocardial regeneration.

Although major advances in genomics have initiated an exciting new era of research, a lack of information regarding cis-regulatory elements has limited the genetic improvement or manipulation of pigs as a meat source and biomedical model. Here, we systematically characterize cis-regulatory elements and their functions in 12 diverse tissues from four pig breeds by adopting similar strategies as the ENCODE and Roadmap Epigenomics projects, which include RNA-seq, ATAC-seq, and ChIP-seq. In total, we generate 199 datasets and identify more than 220,000 cis-regulatory elements in the pig genome. Surprisingly, we find higher conservation of cis-regulatory elements between human and pig genomes than those between human and mouse genomes. Furthermore, the differences of topologically associating domains between the pig and human genomes are associated with morphological evolution of the head and face. Beyond generating a major new benchmark resource for pig epigenetics, our study provides basic comparative epigenetic data relevant to using pigs as models in human biomedical research., To date, little is known about the regulatory landscape of the pig genome. Here, the authors characterize cis-regulatory elements in the pig genome using RNA-seq, ChIP-Seq and ATAC-seq, finding a higher degree of usage conservation between pig and human than mouse and human.

BACKGROUND The blood-brain barrier (BBB) is a major impediment to pharmacological treatment of gliomas. Low-intensity pulsed ultrasound with concomitant administration of intravenous microbubbles (LIPU/MB), temporarily opens the BBB. Here we investigate the pharmacokinetics and safety of this approach of repeated delivery of albumin-bound paclitaxel (Abraxane®, ABX) to the peri-tumoral brain. To perform LIPU/MB-based BBB opening prior to ABX infusions, we used a novel 6 x 6 cm device with 9 ultrasound emitters (SC9) that is implanted in a skull window after tumor resection. METHODS A Phase 1 dose-escalation trial using Bayesian adaptive design was conducted (NCT04528680). Patients with recurrent operable glioblastoma, a WHO PS ≤ 2 and adequate bone marrow and organ function were eligible. After tumor resection and implantation of SC9, repeated cycles of BBB opening by LIPU/MB immediately followed by ABX, was performed every 3 weeks. Intraoperative LIPU/MB and low dose ABX was given prior to tumor resection allowed for investigation tissue concentrations and pharmacokinetics. RESULTS Seventeen patients have been treated at six escalating ABX dose levels (40-260 mg/m2). At dose of 260 mg/m2, a grade 3 reversible taxane-associated dose limiting encephalopathy was observed in one patient. The patient continued treatment at a lower dose in subsequent cycles. A second patient exhibited encephalopathy on cycle 2. One patient developed grade 2 cumulative peripheral neuropathy. Intraoperative pharmacokinetic studies showed that ABX tissue concentrations in non-enhancing peri-tumoral brain were increased 3.7-fold after LIPU/MB. sc-RNA-sequencing showed transcriptional dysregulation of membrane transporters, pathways related to trans-cytosis as well as cell-cell and cell-matrix adhesion. CONCLUSIONS The LIPU/MB using skull-implantable ultrasound enhances the penetration of ABX in large regions of the brain. The procedure can be performed repeatedly and safely. LIPU-based BBB opening leads to transcriptional alterations in brain endothelium. Funding: NIH/NCI 1R01CA245969-01A1, CarThera (SC9 devices), BMS/Celgene (Abraxane®).

Although large-scale and accurate identification of cis-regulatory elements on pig protein-coding and long non-coding genes has been reported, similar study on pig miRNAs is still lacking. Here, we systematically characterized the cis-regulatory elements of pig miRNAs in muscle and fat by adopting miRNAomes, ChIP-seq, ATAC-seq, RNA-seq and Hi-C data. In total, the cis-regulatory elements of 257 (85.95%) expressed miRNAs including 226 known and 31 novel miRNAs were identified. Especially, the miRNAs associated with super-enhancers, active promoters, and "A" compartment were significantly higher than those associated by typical enhancers, prompters without H3K27ac, and "B" compartment, respectively. The tissue specific transcription factors were the primary determination of core miRNA expression pattern in muscle and fat. Moreover, the miRNA promoters are more evolutionarily conserved than miRNA enhancers, like other type genes. Our study adds additional important information to existing pig epigenetic data and provides essential resource for future in-depth investigation of pig epigenetics.

As one of the major cell organelles responsible for ATP production, it is important that neurons maintain mitochondria with structural and functional integrity; this is especially true for neurons with high metabolic requirements. When mitochondrial damage occurs, mitochondria are able to maintain a steady state of functioning through molecular and organellar quality control, thus ensuring neuronal function. And when mitochondrial quality control (MQC) fails, mitochondria mediate apoptosis. An apparently key molecule in MQC is the transcriptional coactivator peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α). Recent findings have demonstrated that upregulation of PGC-1α expression in neurons can modulate MQC to prevent mitochondrial dysfunction in certain in vivo and in vitro aging or neurodegenerative encephalopathy models, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease. Because mitochondrial function and quality control disorders are the basis of pathogenesis in almost all neurodegenerative diseases (NDDs), the role of PGC-1α may make it a viable entry point for the treatment of such diseases. This review focuses on multi-level MQC in neurons, as well as the regulation of MQC by PGC-1α in these major NDDs.

Acute myeloid leukemia (AML) represents a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors, and kinases that can cause epigenetic reshaping. It is unclear to what extent AML mutations drive chromatin 3D structure alteration and contribute to myeloid transformation. We first performed Hi-C and whole-genome sequencing in 25 AML patient samples and seven healthy donor samples, and identified recurrent alterations of A/B compartments, TADs, and chromatin loops that are unique to different subtypes. To investigate how altered chromatin organization contributes to transcriptional misregulation, we performed RNA-Seq, ATAC-Seq and CUT&ag for CTCF, H3K27ac, and H3K27me3 in the same AML samples. We identified extensive and recurrent AML-specific promoter-enhancer and promoter-repressor loops. We performed both CRISPR deletion and interference experiments and validated two repressor loops that downregulated cancer related genes IKZF2 and RTTN. Furthermore, by using our recently developed algorithm, we identified structural variation-induced enhancer-hijacking and repressor-hijacking events in AML samples. We further demonstrated the role of hijacked enhancers in AML cell growth by CRISPR screening, and the role of hijacked repressors by CRISPR de-repression. We performed whole-genome bisulfite sequencing in 20 AML and normal samples, and showed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Finally, by treating the AML cells with the DNA hypomethylating agent and performing triple knockdown of DNMT1/3A/3B, we demonstrated the impact of altered DNA methylation on gene expression and 3D genome organization. Overall this study provides an invaluable resource for leukemia studies and also highlighted the role of repressor-loops and hijacked cis-elements in gene regulation and human diseases. Citation Format: Jie Xu, Fan Song, Baozhen Zhang, Huijue Lyu, Mikoto Kobayashi, Ziyu Zhao, Ye Hou, Xiaotao Wang, Yu Luan, Bei Jia, Lena Stasiak, Qixuan Wang, Qi Jin, Qiushi Jin, Yihao Fu, Ross C. Hardison, Sinisa Dovat, Leonidas C. Platanias, Yue Yang, Tomoko Yamada, Aaron D. Viny, Ross L. Levine, David F. Claxton, James R. Broach, Hong Zheng, Feng Yue. Subtype-specific and structure variation induced 3D genome alteration in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2953.

2016 Background: The blood-brain barrier (BBB) is a major impediment to pharmacological treatment of gliomas, a diffuse tumor infiltrating the peri-tumoral normal brain. Low-intensity pulsed ultrasound directed at the brain with concomitant administration of intravenous microbubbles (LIPU/MB), temporarily opens the BBB. This technique was previously shown with a first generation of the device in combination with carboplatin chemotherapy (Idbaih et al. 2019). Here we investigate the pharmacokinetics and safety of this approach in the context of repeated delivery of albumin-bound paclitaxel (ABX) to the peri-tumoral brain. To perform LIPU/MB-based BBB opening prior to ABX infusions, we used a novel 6 x 6 cm device with 9 ultrasound emitters (SC9) that is implanted in a skull window after tumor resection. Methods: A Phase 1 dose-escalation trial using Bayesian adaptive design was initiated at our institution (NCT04528680). Patients with recurrent operable glioblastoma, a WHO PS ≤ 2 and normal bone marrow and organ function were eligible. After tumor resection and implantation of SC9, repeated cycles of BBB opening by LIPU/MB immediately followed by ABX, was performed every 3 weeks. Intraoperative LIPU/MB and low dose ABX was given prior to tumor resection for investigation of pharmacokinetics. Results: Seventeen patients have been enrolled and six dose levels of ABX were used (40-260 mg/m2). Severe, reversible taxane-associated encephalopathy was observed in one patient at the max. planned dose level (260 mg/m2). The patient continued treatment at a lower dose in subsequent cycles. One patient developed grade 2 cumulative peripheral neuropathy. Other mild to moderate and reversible toxicities for ABX including myelosuppression, fatigue, alopecia were observed as expected. Intraoperative sonication and pharmacokinetic studies showed that ABX tissue concentrations in non-enhancing peri-tumoral brain were increased several-fold after LIPU/MB. On electron microscopy, sonicated tissue showed ultra-structural alterations in brain capillary endothelial cells. Molecular studies showed transcriptional dysregulation of membrane transporters, pathways related to trans-cytosis, cell permeability as well as cell-cell and cell-matrix adhesion. Updated results will be presented. Conclusions: The LIPU/MB using skull-implantable ultrasound enhances the penetration of large chemotherapeutic drugs such as ABX in large regions of the brain, a procedure that can be performed repeatedly and safely. LIPU-based BBB opening leads to ultrastructural and transcriptional alterations in brain endothelial cells. A Phase 2 clinical trial is planned to investigate efficacy of this approach. Funding: NIH/NCI 1R01CA245969-01A1, Carthera (SC9 devices), Celgene/BMS, Malnati Brain Tumor Institute, Moceri Family Foundation. Clinical trial information: NCT04528680.

Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown.ResultWe determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration.ConclusionIn summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are morbid brain tumors. Even with treatment survival is poor, making pHGG the number one cause of cancer death in children. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding histone H3. To investigate whether H3K27M is associated with distinct chromatin structure that alters transcription regulation, we generated the first high-resolution Hi-C maps of pHGG cell lines and tumor tissue. By integrating transcriptome (RNA-seq), enhancer landscape (ChIP-seq), genome structure (Hi-C), and chromatin accessibility (ATAC-seq) datasets from H3K27M and wild-type specimens, we identified tumor-specific enhancers and regulatory networks for known oncogenes. We identified genomic structural variations that lead to potential enhancer hijacking and gene coamplification, including A2M, JAG2, and FLRT1 Together, our results imply three-dimensional genome alterations may play a critical role in the pHGG epigenetic landscape and contribute to tumorigenesis.

The purpose of this research is to develop a liposomal drug delivery system, which can selectively target hepatocellular carcinoma (HCC) to deliver the antitumor agent N-14NCTDA, a C14 alkyl chain norcantharimide derivative of norcantharidin. N-14NCTDA-loaded liposomes were successfully prepared by lipid membrane hydration and extrusion methods. SP94, a targeting peptide for HCC cells, was attached to the liposomes loaded with N-14NCTDA by the post-insertion method to obtain SP94 modified liposomes (SP94-LPs). SP94-LPs had a significant cytotoxicity against Hep G2 cells with the IC50 of 15.395 ± 0.89 μg/mL, which is lower than that of NCTD-S (IC50 = 20.863 ± 0.56 μg/mL) and GAL-LPs (IC50 = 24.589 ± 1.02 μg/mL). Compared with conventional liposomes (Con-LPs), SP94-LPs showed greater cellular uptake in Hep G2 cells. Likewise, significant tumor suppression was achieved in H22 tumor-bearing mice which were treated with SP94-LPs. The tumor inhibition rate (IRw) of SP94-LPs was 82 ± 0.98%, obviously higher than that of GAL-LPs (69 ± 1.39%), Con-LPs (60 ± 2.78%), and NCTD-S (51 ± 3.67%). SP94-LPs exhibited a significant hepatocellular carcinoma-targeting activity in vitro and in vivo, which will provide a new alternative for hepatocellular carcinoma treatment in future.

miRNAs reportedly participate in various biological processes, such as skeletal muscle proliferation and differentiation. However, the regulation of differentially expressed (DE) miRNAs and their function in myogenesis remain unclear. Herein, miRNA expression profiles and regulation during C2C12 differentiation were analyzed in relation to chromatin states by RNA-seq, ATAC-seq, and ChIP-seq. We identified 19 known and nine novel differentially expressed miRNAs at days 0, 1, 2, and 4. The expression of the differentially expressed miRNAs was related to the chromatin states of the 113 surrounding open chromatin regions defined by ATAC-seq peaks. Of these open chromatin regions, 44.25% were colocalized with MyoD/MyoG binding sites. The remainder of the above open chromatin regions were enriched with motifs of the myoblast-expressed AP-1 family, Ctcf, and Bach2 transcription factors (TFs). Additionally, the target genes of the above differentially expressed miRNAs were enriched primarily in muscle growth and development pathways, especially the Hippo signaling pathway. Moreover, via combining a loss-of-function assay with Q-PCR, western blotting, and immunofluorescence, we confirmed that the Hippo signaling pathway was responsible for C2C12 myoblast differentiation. Thus, our results showed that these differentially expressed miRNAs were regulated by chromatin states and affected muscle differentiation through the Hippo signaling pathway. Our findings provide new insights into the function of these differentially expressed miRNAs and the regulation of their expression during myoblast differentiation.

Objective This study aims to clarify the mechanisms underlying transcriptional regulation and regulatory roles of lncRNAs in skeletal muscle cell differentiation. Methods We analysed the expression patterns of lncRNAs via time‐course RNA‐seq. Then, we further combined the ATAC‐seq and ChIP‐seq to investigate the governing mechanisms of transcriptional regulation of differentially expressed (DE) lncRNAs. Weighted correlation network analysis and GO analysis were conducted to identify the transcription factor (TF)‐lncRNA pairs related to skeletal muscle cell differentiation. Results We identified 385 DE lncRNAs during C2C12 differentiation, the transcription of which is determined by chromatin states around their transcriptional start sites. The TF‐lncRNA correlation network showed substantially concordant changes in DE lncRNAs between C2C12 differentiation and satellite cell rapid growth stages. Moreover, the up‐regulated lncRNAs showed a significant decrease following the differentiation capacity of satellite cells, which gradually declines during skeletal muscle development. Notably, inhibition of the lncRNA Atcayos and Trp53cor1 led to the delayed differentiation of satellite cells. Those lncRNAs were significantly up‐regulated during the rapid growth stage of satellite cells (4‐6 weeks) and down‐regulated with reduced differentiation capacity (8‐12 weeks). It confirms that these lncRNAs are positively associated with myogenic differentiation of satellite cells during skeletal muscle development. Conclusions This study extends the understanding of mechanisms governing transcriptional regulation of lncRNAs and provides a foundation for exploring their functions in skeletal muscle cell differentiation., Schematic of TF (MyoD, MyoG, Mef2c and Heyl) and lncRNA (Atcayos and Trp53cor1) regulatory interactions in differentiating C2C12 and ageing satellite cells.

c-Myc (Myc hereafter) is found to be deregulated and/or amplified in most acute myeloid leukemias (AMLs). Almost all AML cells are dependent upon Myc for their proliferation and survival. Thus, Myc has been proposed as a critical anti-AML target. Myc has Max-mediated transactivational and Myc-interacting zinc finger protein 1 (Miz1)-mediated transrepressional activities. The role of Myc-Max–mediated transactivation in the pathogenesis of AML has been well studied; however, the role of Myc-Miz1–mediated transrepression in AML is still somewhat obscure. Myc protein harboring a V394D mutation (Myc(V394D)) is a mutant form of Myc that lacks transrepressional activity due to a defect in its ability to interact with Miz1. We found that, compared with Myc, the oncogenic function of Myc(V394D) is significantly impaired. The AML/myeloproliferative disorder that develops in mice receiving Myc(V394D)-transduced hematopoietic stem/progenitor cells (HSPCs) is significantly delayed compared with mice receiving Myc-transduced HSPCs. Using a murine MLL-AF9 AML model, we found that AML cells expressing Myc(V394D) (intrinsic Myc deleted) are partially differentiated and show reductions in both colony-forming ability in vitro and leukemogenic capacity in vivo. The reduced frequency of leukemia stem cells (LSCs) among Myc(V394D)-AML cells and their reduced leukemogenic capacity during serial transplantation suggest that Myc-Miz1 interaction is required for the self-renewal of LSCs. In addition, we found that Myc(V394D)-AML cells are more sensitive to chemotherapy than are Myc-AML cells. Mechanistically, we found that Myc represses Miz1-mediated expression of CCAAT/enhancer-binding protein α (Cebpα) and Cebpδ, thus playing an important role in the pathogenesis of AML by maintaining the undifferentiated state and self-renewal capacity of LSCs.

Transfusion of autologous late‐outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human‐, rabbit‐ and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium‐2 as the standard culture medium while exploring the mechanisms used endothelial basal medium‐2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H2O2 were quantified with DAF‐FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H2O2 assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration‐dependently enhanced OEC number increase in vitro. Transfusion of bilobalide‐based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L‐NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration‐dependently enhanced the eNOS Ser‐1177 phosphorylation and Thr‐495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H2O2 in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide‐based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous‐OECs‐transfusion therapy.

Nuclear condensation and enucleation are characteristic processes in mammalian terminal erythropoiesis. These processes are associated with the transient nuclear opening formation that mediates partial histone release to the cytoplasm. Our previous report showed that caspases are involved in the cleavage of nuclear lamina to enable histone release. However, it remains unclear whether nuclear opening formation and histone release regulate the genomic three-dimensional organization during nuclear condensation. To answer this question, we cultured E13.5 mouse fetal liver Ter119 negative erythroid progenitor cells in erythropoietin (EPO) containing medium for 48 h with or without the presence of caspase inhibitor. As expected, caspase inhibitor blocked nuclear opening formation and histone release, and significantly reduced nuclear condensation and enucleation. We next performed a Hi-C sequencing to investigate chromatin structural change during terminal differentiation and nuclear condensation. To this end, the cultured fetal liver erythroid cells with or without caspase inhibitor were harvested at 30 h right before enucleation for Hi-C sequencing. The sequencing results showed that cells at 30 h contain significantly more interactions than freshly isolated erythroid progenitors, which is consistent with chromatin condensation during terminal erythropoiesis. Further analysis showed that increased interactions mainly accumulate as inter-chromosomal interactions, suggesting inter-chromosome interaction is the dominant structural force driving erythrocyte chromatin condensation. Surprisingly, there were no significant chromatin structural changes between caspase inhibitor treated and mock-treated cells when compared at 30 h. We also performed ATAC-seq and RNA-seq with the same experiment settings, both corresponded to Hi-C sequencing and showed little difference under caspase inhibitor treatment. These results indicate that although histone release and nuclear condensation are compromised with the inhibition of caspases, chromatin stays condensed with well-organized three-dimensional structure and appropriate gene expression regulations. To further confirm this phenomenon, we generated caspase-3 and -7 double knock out (cas3cas7-/-) mice. Cas3cas7-/- mice are embryonically lethal due to defective cardiac development. The hematopoietic tissues in these mice have not been well studied. We harvested fetal liver Ter119 negative erythroid progenitor cells from E13.5 cas3cas7-/- mice and the cells from the littermate (cas7-/-, cas3+/-cas7-/-) mice were used as controls. We first cultured Ter119 negative fetal liver erythroid progenitors in EPO containing medium for 48 h. Immunofluorescence analysis showed that the nuclear opening was significantly inhibited, and the nuclear size significantly increased in the erythroid cells from cas3cas7-/- mice due to failure of histone release into cytoplasm. Flow cytometry analysis showed that enucleation was significantly impaired in cas3cas7-/- cells, but the cells could still differentiate although with lower efficiency. We further performed an in vivo assay in which E13.5 cas3cas7-/- fetal liver cells were transplanted into wild type lethally irradiated recipient mice. EPO medium cultured bone marrow lineage negative cells from these transplanted mice showed significant reduction in nuclear opening and histone release, and enlargement of nuclear size. However, these mice survived well despite anemia. These results indicate a portion of orthochromatic erythroblasts managed to enucleate even with the less condensed nuclei. Overall, our study demonstrates that nuclear opening and histone release are essential for nuclear condensation but have minimal effects on chromatin condensation or the regulation of gene expression in terminal erythropoiesis. Appropriate nuclear condensation is important for efficient enucleation. However, orthochromatic erythroblasts could still manage to enucleate although with low efficacies. Disclosures No relevant conflicts of interest to declare.

Urinary bladder cancers (BLCA) are commonly diagnosed urologic malignancy in the United States. Muscle invasive BLCA is a morbid and expensive disease to treat, thus increased molecular understanding is necessary. BLCA has recently been characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARg have been shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remains unknown. Here, we determined the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles (FOXA1 and GATA3) in luminal and basal subtypes of bladder cancers. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct TF binding at distal-enhancers of luminal and basal bladder cancers. Finally, we identified a novel clinically relevant transcriptional factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. In summary, our work identifies a subtype-specific epigenomic and 3D genome structure in urinary bladder cancers and suggests a novel link between the circadian TF NPAS2 and a clinical bladder cancer subtype. Citation Format: Tejaswi Tejaswi, Baozhen Zhang, Qixuan Wang, Ye Hou, Qiushi Jin, Jie Xu, Hongbo Yang, Tingting Liu, David J. DeGraff, Feng Yue. Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2189.