KLF4 inhibits early neural differentiation of ESCs by coordinating specific 3D chromatin structure.

Neural differentiation of embryonic stem cells (ESCs) requires precisely orchestrated gene regulation, a process governed in part by changes in 3D chromatin structure. How these changes regulate gene expression in this context remains unclear. In this study, we observed enrichment of the transcription factor KLF4 at some poised or closed enhancers at TSS-linked regions of genes associated with neural differentiation. Combination analysis of ChIP, HiChIP and RNA-seq data indicated that KLF4 loss in ESCs induced changes in 3D chromatin structure, including increased chromatin interaction loops between neural differentiation-associated genes and active enhancers, leading to upregulated expression of neural differentiation-associated genes and therefore early neural differentiation. This study suggests KLF4 inhibits early neural differentiation by regulation of 3D chromatin structure, which is a new mechanism of early neural differentiation.

Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs.

Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1-Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis.

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA.

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer.

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer-promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.

HOTAIRM1, an enhancer lncRNA, promotes glioma proliferation by regulating long-range chromatin interactions within HOXA cluster genes.

The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.

Bivalent activity of super-enhancer RNA LINC02454 controls 3D chromatin structure and regulates glioma sensitivity to temozolomide.

Glioma cell sensitivity to temozolomide (TMZ) is critical for effective treatment and correlates with patient survival, although mechanisms underlying this activity are unclear. Here, we reveal a new mechanism used by glioma cells to modulate TMZ sensitivity via regulation of SORBS2 and DDR1 genes by super-enhancer RNA LINC02454. We report that LINC02454 activity increases glioma cell TMZ sensitivity by maintaining long-range chromatin interactions between SORBS2 and the LINC02454 enhancer. By contrast, LINC02454 activity also decreased glioma cell TMZ sensitivity by promoting DDR1 expression. Our study suggests a bivalent function for super-enhancer RNA LINC02454 in regulating glioma cell sensitivity to TMZ.

Two Enhancers Regulate HoxB Genes Expression During Retinoic Acid-Induced Early Embryonic Stem Cells Differentiation Through Long-Range Chromatin Interactions.

Homeobox B cluster (HoxB) genes play important roles in retinoic acid (RA)-induced early embryonic stem cells (ESCs) differentiation. Knowledge of regulation network of HoxB is important to further unveil the mechanism of ESCs differentiation. In this study, we identified two enhancers that were activated by RA treatment and 4C data showed long-range interactions between HoxB genes and the two enhancers. CRISPR/Cas9-mediated individual or compound deletion of the two enhancers significantly inhibits HoxB gene expression, and transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in the enhancer KO cells. We propose new mechanism by which two enhancers regulate HoxB gene expression by different regulation modes during RA-induced early ESCs differentiation through long-range chromatin interactions.

lncRNA 5430416N02Rik Promotes the Proliferation of Mouse Embryonic Stem Cells by Activating Mid1 Expression through 3D Chromatin Architecture.

Both 3D chromatin architecture and long non-coding RNAs (lncRNAs) play essential roles in pluripotency maintenance. However, whether lncRNAs are involved in organizing 3D chromatin structure remains largely unexplored. We identified 39 lncRNAs bound by Klf4, among which we further revealed the 5430416N02Rik promoter is a chromatin interaction hub. Knockout of the 5430416N02Rik locus reduces the proliferation rate of embryonic stem cells (ESCs). Moreover, deleting both the promoter and the gene body of 5430416N02Rik causes a more severe proliferation defect and has a more profound impact on the transcriptome than deleting the gene body alone. The reduced proliferation of the 5430416N02Rik locus knockout ESCs is mainly due to the downregulation of Mid1, the expression of which requires the inter-chromosomal interaction between Mid1 and 5430416N02Rik loci. In summary, our data demonstrated that the lncRNA 5430416N02Rik gene locus maintains the fast proliferation of ESCs by activating the expression of Mid1 through chromatin interaction.

Branched-chain amino acid aminotransferase-1 regulates self-renewal and pluripotency of mouse embryonic stem cells through Ras signaling.

The developmental plasticity of embryonic stem cells (ESCs) is mainly controlled by well-characterized transcription factors, but additional factors, especially those related to metabolism that modulate this intrinsic program remain elusive. Here, using whole transcriptome analysis, we identified branched-chain amino acid aminotransferase-1(Bcat1) as highly-expressed in mouse ESCs and dramatically down-regulated upon differentiation. Bcat1 deletion impaired pluripotency and self-renewal in mouse ESCs, while Bcat1 overexpression resulted in robust ESC self-renewal and inhibition of differentiation. Whole genome bisulfite sequencing (WGBS) analysis showed that Bcat1 deletion altered whole genome methylation levels and hence gene expression in multiple pathways. Specifically, Bcat1 deletion increased expression of RAS protein activator like 1(Rasal1), leading to inactivation of Ras-Erk/MAPK signaling, while Rasal1 inhibition rescued defects seen in Bcat1 deleted cells. In summary, we demonstrate that Bcat1 is essential for mouse ESC self-renewal and pluripotency and that this effect is mediated by DNA methylation and the Ras signaling pathway.

BCL11A expression in acute phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) has chronic and acute phases. In chronic phase myeloid differentiation is preserved whereas in acute phase myeloid differentiation is blocked. Acute phase CML resembles acute myeloid leukemia (AML). Chronic phase CML is caused by BCR-ABL1. What additional mutation(s) cause transition to acute phase is unknown and may differ in different persons with CML. BCL11A encodes a transcription factor and is aberrantly-expressed in several haematological and solid neoplasms. We analyzed BCL11A mRNA levels in subjects with chronic and acute phase CML. BCL11A transcript levels were increased in subjects with CML in acute phase compared with those in normals and in subjects in chronic phase including some subjects studied in both phases. BCL11A mRNA levels were correlated with percent bone marrow blasts and significantly higher in lymphoid versus myeloid blast crisis. Differentiation of K562 with butyric acid, a CML cell line, decreased BCL11A mRNA levels. Cytology and flow cytometry analyses showed that ectopic expression of BCL11A in K562 cells blocked differentiation. These data suggest BCL11A may operate in transformation of CML from chronic to acute phase in some persons.

BCL11A expression in acute myeloid leukemia.

BACKGROUND: BCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. OBJECTIVE: Interrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. METHODS: We determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. RESULTS: Subjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th-75th inter-quartile range, 1.33-4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3A(R882) and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P=0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P=0.35) in the high BCL11A cohort. CONCLUSION: BCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.

Aberrant expression of ecotropic viral integration site-1 in acute myeloid leukemia and acute lymphoblastic leukemia.

Ecotropic viral integration site-1 (EVI1) proto-oncogene expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) requires further investigation. Here, EVI1 expression levels were measured in 216 Chinese patients with AML and 67 with ALL via quantitative real-time polymerase chain reaction. We found that EVI1 expressed at a high level (H-EVI1) was present in 11.1% of patients with AML versus 20.9% with ALL. Low levels of EVI1 expression occurred in 23.1% with AML versus 43.3% with ALL. This suggested that alteration of EVI1 expression was more profound in ALL than in AML. H-EVI1 was significantly enriched in 30-60-year-old patients. French-American-British (FAB) M3 subtype was significantly correlated with H-EVI1. Interestingly, we found that EVI1 expression was negatively associated with presence of the Philadelphia chromosome (Ph+) and MLL rearrangements in AML. However, Ph+, but not MLL rearrangements, was inversely correlated with EVI1 expression in B-ALL. These results for the first time suggest a mutually exclusive relationship between EVI1 expression and Ph+ karyotype.

Low frequency of mutations in Chinese with acute myeloid leukemia: Different disease or different aetiology?

Mutations in FLT3, DNMT3A, NRAS, NF1 and TP53 occur in persons of predominately European descent with acute myeloid leukemia (AML). Some, such as internal tandem duplication of FLT3 (FLT3-ITD) and point mutations in DNMT3A and NRAS, are especially frequent whereas others such as NF1 and TP53 are less so. Frequencies of these mutations in persons with seemingly similar AML from other genetic groups are largely unknown. We studied 269 Chinese (mostly Han) with de novo AML. FLT3-ITD was detected in 51 subjects (23%; 95% CI, 17-28%), R882 mutation of DNMT3A in 17 (6%; 95% CI, 3-9%) and NRAS mutation in 17 (7%; 95% CI, 3-9%). No mutations in NF1 and only 1 mutation in TP53 (1%, 95% CI, <2.5%) were detected. Except for FLT3-ITD, frequencies of these mutations are significantly less than those in persons of predominately European descent with AML. The reason(s) for this disparity is unknown but may offer clues to the aetiology of AML in different populations or may indicate some mutations associated with AML in persons of predominately European descent are not fundamental to the aetiology of the disease.

Cellular intrinsic mechanism affecting the outcome of AML treated with Ara-C in a syngeneic mouse model.

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.