LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade.

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

Set2 regulates Ccp1 and Swc2 to ensure centromeric stability by retargeting CENP-A.

Precise positioning of the histone-H3 variant, CENP-A, ensures centromere stability and faithful chromosomal segregation. Mislocalization of CENP-A to extra-centromeric loci results in aneuploidy and compromised cell viability associated with formation of ectopic kinetochores. The mechanism that retargets mislocalized CENP-A back to the centromere is unclarified. We show here that the downregulation of the histone H3 lysine 36 (H3K36) methyltransferase Set2 can preserve centromere localization of a temperature-sensitive mutant cnp1-1 Schizosaccharomyces pombe CENP-A (SpCENP-A) protein and reverse aneuploidy by redirecting mislocalized SpCENP-A back to centromere from ribosomal DNA (rDNA) loci, which serves as a sink for the delocalized SpCENP-A. Downregulation of set2 augments Swc2 (SWR1 complex DNA-binding module) expression and releases histone chaperone Ccp1 from the centromeric reservoir. Swc2 and Ccp1 are directed to the rDNA locus to excavate the SpCENP-Acnp1-1, which is relocalized to the centromere in a manner dependent on canonical SpCENP-A loaders, including Mis16, Mis17 and Mis18, thereby conferring cell survival and safeguarding chromosome segregation fidelity. Chromosome missegregation is a severe genetic instability event that compromises cell viability. This mechanism thus promotes CENP-A presence at the centromere to maintain genomic stability.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis.

Adenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research.

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.

Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances.

BACKGROUND: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance. METHODS: Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS). Archival SurePlex-amplified trophectoderm biopsy samples (n = 96) previously analyzed using the commercial kit were blinded and reanalyzed using Nanopore. RESULTS: Nanopore-based PGT-A identified the specific aberration in 95.45% (84/88) and 97.78% (88/90) of single-/multicells with an aneuploidy or segmental imbalance (10-30.5 Mb), respectively. Comparison against the commercial kit's results revealed concordances of 98.86% (87/88) and 98.89% (89/90) for the aneuploid and segmentally unbalanced (10-30.5 Mb aberration) samples, respectively. Detection sensitivity for smaller segmental imbalances (5-5.8 Mb aberration, n = 30) decreased markedly on both platforms. Nanopore-based PGT-A reanalysis of trophectoderm biopsy samples was 97.92% (94/96) concordant with the commercial kit results. CONCLUSION: Up to 24 SurePlex-amplified single-cell, multicell, or trophectoderm samples could be sequenced in a single MinION flow-cell for subsequent preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) analysis, with results obtainable in ≤3 days and at per-sample costs that are competitive with commercial offerings. Nanopore's third-generation single-molecule sequencing represents a viable alternative to current commercial NGS-based PGT-A solutions for aneuploidy and segmental imbalance (≥10 Mb) screening of single-/multicell or trophectoderm biopsy samples.

Myeloid lncRNA LOUP mediates opposing regulatory effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here, we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA and DNA interactions with the broadly expressed Runt-related transcription factor 1 (RUNX1), we identified the long noncoding RNA (lncRNA) originating from the upstream regulatory element of PU.1 (LOUP). This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia (AML), wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein, RUNX1-ETO, limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell-type-specific RNAs and transcription factors, as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.

A Runx1-enhancer Element eR1 Identified Lineage Restricted Mammary Luminal Stem Cells.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli-eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Molecular profiling reveals a tumor-promoting phenotype of monocytes and macrophages in human cancer progression.

Monocytes and macrophages are major components of the tumor microenvironment, but their contributions to human cancer are poorly understood. We used molecular profiling combined with functional assays to investigate the role of these cells in human renal cell carcinoma (RCC). Blood monocytes from RCC patients displayed a tumor-promoting transcriptional profile that supported functions like angiogenesis and invasion. Induction of this protumor phenotype required an interleukin-1 receptor (IL-1R)-dependent mechanism. Indeed, targeting of IL-1-IL-1R axis in a human RCC xenograft model abrogated the protumor phenotype of tumor-associated macrophages (TAMs) and reduced tumor growth in vivo. Supporting this, meta-analysis of gene expression from human RCC tumors showed IL1B expression to correlate with myelomonocytic markers, protumor genes, and tumor staging. Analyzing RCC patient tumors confirmed the protumor phenotype of TAMs. These data provide direct evidence for a tumor-promoting role of monocytes and macrophages in human cancer and indicate IL-1-IL-1R as a possible therapeutic target.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.

ZRSR1 co-operates with ZRSR2 in regulating splicing of U12-type introns in murine hematopoietic cells.

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.

CD8+ mycosis fungoides with ichthyosiform clinical presentation and angiocentric feature.

Mycosis fungoides (MF) is characterized by epidermotropic atypical lymphocytes infiltrate with α/ß T-helper memory immunophenotype (ßF1+, CD3+, CD4+, CD45Ro+, and CD8-). Angiocentricity is always associated with aggressive behavior or poor outcome in primary or secondary cutaneous lymphomas. Rare cases of angiocentric MF with a T-cytotoxic immunophenotype (CD3+, CD4-, CD8+, TIA-1+) have been described. Here, we report a 27-year-old man diagnosed with MF, clinically presenting with ichthyosiform lesions on his trunk and limbs. Biopsy demonstrated a CD3+ and CD8+ atypical lymphocytic infiltrate with marked epidermotropism and angiocentricity. Awareness of this rare MF variant with unusual clinicopathological characteristics is important to avoid misdiagnosis.

Dynamic Phase Extraction: Applications in Pulse Rate Variability.

Pulse rate variability is a physiological parameter that has been extensively studied and correlated with many physical ailments. However, the phase relationship between inter-beat interval, IBI, and breathing has very rarely been studied. Develop a technique by which the phase relationship between IBI and breathing can be accurately and efficiently extracted from photoplethysmography (PPG) data. A program based on Lock-in Amplifier technology was written in Python to implement a novel technique, Dynamic Phase Extraction. It was tested using a breath pacer and a PPG sensor on 6 subjects who followed a breath pacer at varied breathing rates. The data were then analyzed using both traditional methods and the novel technique (Dynamic Phase Extraction) utilizing a breath pacer. Pulse data was extracted using a PPG sensor. Dynamic Phase Extraction (DPE) gave the magnitudes of the variation in IBI associated with breathing [Formula: see text] measured with photoplethysmography during paced breathing (with premature ventricular contractions, abnormal arrhythmias, and other artifacts edited out). [Formula: see text] correlated well with two standard measures of pulse rate variability: the Standard Deviation of the inter-beat interval (SDNN) (ρ = 0.911) and with the integrated value of the Power Spectral Density between 0.04 and 0.15 Hz (Low Frequency Power or LF Power) (ρ = 0.885). These correlations were comparable to the correlation between the SDNN and the LF Power (ρ = 0.877). In addition to the magnitude [Formula: see text], Dynamic Phase Extraction also gave the phase between the breath pacer and the changes in the inter-beat interval (IBI) due to respiratory sinus arrythmia (RSA), and correlated well with the phase extracted using a Fourier transform (ρ = 0.857). Dynamic Phase Extraction can extract both the phase between the breath pacer and the changes in IBI due to the respiratory sinus arrhythmia component of pulse rate variability ([Formula: see text], but is limited by needing a breath pacer.

Exchange Coupling in a Linear Chain of Three Quantum-Dot Spin Qubits in Silicon.

Quantum gates between spin qubits can be implemented leveraging the natural Heisenberg exchange interaction between two electrons in contact with each other. This interaction is controllable by electrically tailoring the overlap between electronic wave functions in quantum dot systems, as long as they occupy neighboring dots. An alternative route is the exploration of superexchange-the coupling between remote spins mediated by a third idle electron that bridges the distance between quantum dots. We experimentally demonstrate direct exchange coupling and provide evidence for second neighbor mediated superexchange in a linear array of three single-electron spin qubits in silicon, inferred from the electron spin resonance frequency spectra. We confirm theoretically, through atomistic modeling, that the device geometry only allows for sizable direct exchange coupling for neighboring dots, while next-nearest neighbor coupling cannot stem from the vanishingly small tail of the electronic wave function of the remote dots, and is only possible if mediated.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

RNA editing mediates the functional switch of COPA in a novel mechanism of hepatocarcinogenesis.

BACKGROUND & AIMS: RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant adenosine-to-inosine RNA editing is implicated in cancers. Until now, very few functionally important protein-recoding editing targets have been discovered. Here, we investigated the role of a recently discovered protein-recoding editing target COPA (coatomer subunit α) in hepatocellular carcinoma (HCC). METHODS: Clinical implication of COPA editing was studied in a cohort of 125 HCC patients. CRISPR/Cas9-mediated knockout of the editing site complementary sequence (ECS) was used to delete edited COPA transcripts endogenously. COPA editing-mediated change in its transcript or protein stability was investigated upon actinomycin D or cycloheximide treatment, respectively. Functional difference in tumourigenesis between wild-type and edited COPA (COPAWTvs. COPAI164V) and the exact mechanisms were also studied in cell models and mice. RESULTS: ADAR2 binds to double-stranded RNA formed between edited exon 6 and the ECS at intron 6 of COPA pre-mRNA, causing an isoleucine-to-valine substitution at residue 164. Reduced editing of COPA is implicated in the pathogenesis of HCC, and more importantly, it may be involved in many cancer types. Upon editing, COPAWT switches from a tumour-promoting gene to a tumour suppressor that has a dominant-negative effect. Moreover, COPAI164V may undergo protein conformational change and therefore become less stable than COPAWT. Mechanistically, COPAI164V may deactivate the PI3K/AKT/mTOR pathway through downregulation of caveolin-1 (CAV1). CONCLUSIONS: We uncover an RNA editing-associated mechanism of hepatocarcinogenesis by which downregulation of ADAR2 caused the loss of tumour suppressive COPAI164V and concurrent accumulation of tumour-promoting COPAWT in tumours; a rapid degradation of COPAI164V protein and hyper-activation of the PI3K/AKT/mTOR pathway further promote tumourigenesis. LAY SUMMARY: RNA editing is a process in which RNA is changed after it is made from DNA, resulting in an altered gene product. In this study, we found that RNA editing of a gene known as coatomer subunit α (COPA) is lower in tumour samples and discovered that this editing process changes COPA protein from a tumour-promoting form to a tumour-suppressive form. Loss of the edited COPA promotes the development of liver cancer.

dbInDel: a database of enhancer-associated insertion and deletion variants by analysis of H3K27ac ChIP-Seq.

SUMMARY: Cancer hallmarks rely on its specific transcriptional programs, which are dysregulated by multiple mechanisms, including genomic aberrations in the DNA regulatory regions. Genome-wide association studies have shown many variants are found within putative enhancer elements. To provide insights into the regulatory role of enhancer-associated non-coding variants in cancer epigenome, and to facilitate the identification of functional non-coding mutations, we present dbInDel, a database where we have comprehensively analyzed enhancer-associated insertion and deletion variants for both human and murine samples using ChIP-Seq data. Moreover, we provide the identification and visualization of upstream TF binding motifs in InDel-containing enhancers. Downstream target genes are also predicted and analyzed in the context of cancer biology. The dbInDel database promotes the investigation of functional contributions of non-coding variants in cancer epigenome. AVAILABILITY AND IMPLEMENTATION: The database, dbInDel, can be accessed from http://enhancer-indel.cam-su.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of a novel enhancer of CEBPE essential for granulocytic differentiation.

CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.