HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

The mRNA-binding protein IGF2BP1 maintains intestinal barrier function by up-regulating occludin expression.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate-induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis.

Transcriptomic analysis of ileal tissue from Crohn's disease patients identifies extracellular matrix genes that distinguish individuals by age at diagnosis.

Crohn's disease (CD) is a debilitating gastrointestinal (GI) disorder that can impact the entirety of the GI tract. While substantial progress has been made in the medical management of CD, it remains incurable, frequently relapses, and is a significant financial and medical burden. The pathophysiology of CD is not well understood, but it is thought to arise in genetically susceptible individuals upon an environmental insult. Further elucidation of the disease etiology promises to expose additional therapeutic avenues, with the hope of reducing the burden of CD. One approach to understanding disease pathophysiology is to identify clinically relevant molecular disease subsets by using transcriptomics. In this report, we use hierarchical clustering of the ileal transcriptomes of 34 patients and identify two CD subsets. Clinically, these clusters differed in the age of the patients at CD diagnosis, suggesting that age of onset affects disease pathophysiology. The clusters were segregated by three major gene ontology categories: developmental processes, ion homeostasis, and the immune response. Of the genes constituting the immune system category, expression of extracellular matrix-associated genes, COL4A1, S100A9, ADAMTS2, SERPINE1, and FCN1, exhibits the strongest correlation with an individual's age at CD diagnosis. Together these findings demonstrate that transcriptional profiling is a powerful approach to subclassify CD patients.

Identification of a rare LAMB4 variant associated with familial diverticulitis through exome sequencing.

Diverticulitis is a chronic disease of the colon in which diverticuli, or outpouching through the colonic wall, become inflamed. Although recent observations suggest that genetic factors may play a significant role in diverticulitis, few genes have yet been implicated in disease pathogenesis and familial cases are uncommon. Here, we report results of whole exome sequencing performed on members from a single multi-generational family with early onset diverticulitis in order to identify a genetic component of the disease. We identified a rare single nucleotide variant in the laminin ß 4 gene (LAMB4) that segregated with disease in a dominant pattern and causes a damaging missense substitution (D435N). Targeted sequencing of LAMB4 in 148 non-familial and unrelated sporadic diverticulitis patients identified two additional rare variants in the gene. Immunohistochemistry indicated that LAMB4 localizes to the myenteric plexus of colonic tissue and patients harboring LAMB4 variants exhibited reduced LAMB4 protein levels relative to controls. Laminins are constituents of the extracellular matrix and play a major role in regulating the development and function of the enteric nervous system. Reduced LAMB4 levels may therefore alter innervation and morphology of the enteric nervous system, which may contribute to colonic dysmotility associated with diverticulitis.

TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells.

The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics and cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and ß-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs.

TCF7L1 regulates colorectal cancer cell migration by repressing GAS1 expression.

Dysregulated Wnt/ß-catenin signaling is a common feature of colorectal cancer (CRC). The T-cell factor/lymphoid enhancer factor (TCF/LEF; hereafter, TCF) family of transcription factors are critical regulators of Wnt/ß-catenin target gene expression. Of the four TCF family members, TCF7L1 predominantly functions as a transcriptional repressor. Although TCF7L1 has been ascribed an oncogenic role in CRC, only a few target genes whose expression it regulates have been characterized in this cancer. Through transcriptome analyses of TCF7L1 regulated genes, we noted enrichment for those associated with cellular migration. By silencing and overexpressing TCF7L1 in CRC cell lines, we demonstrated that TCF7L1 promoted migration, invasion, and adhesion. We localized TCF7L1 binding across the CRC genome and overlapped enriched regions with transcriptome data to identify candidate target genes. The growth arrest-specific 1 (GAS1) gene was among these and we demonstrated that GAS1 is a critical mediator of TCF7L1-dependent CRC cell migratory phenotypes. Together, these findings uncover a novel role for TCF7L1 in repressing GAS1 expression to enhance migration and invasion of CRC cells.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting FAS signaling.

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, whereas long noncoding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between HOTTIP lncRNA and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-seq (HOTTIP binding in genomewide), ChIP-seq, and ATAC-seq, we found that some miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a, and miR-10b), located cis and trans, were most dramatically regulated and significantly decreased in HOTTIP-/- AML cells. HOTTIP bound to the miR-196b promoter and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster-associated miRNAs in AML cells, whereas reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival of mice compared to wild-type cells transplanted into mice. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription and consequently represses tumor suppressors and promotes leukemogenesis.

Transcriptome Analyses Identify Deregulated MYC in Early Onset Colorectal Cancer.

Despite a global decrease in colorectal cancer (CRC) incidence, the prevalence of early-onset colorectal cancer (EOCRC), or those occurring in individuals before the age of 50, has steadily increased over the past several decades. When compared to later onset colorectal cancer (LOCRC) in individuals over 50, our understanding of the genetic and molecular underpinnings of EOCRCs is limited. Here, we conducted transcriptomic analyses of patient-matched normal colonic segments and tumors to identify gene expression programs involved in carcinogenesis. Amongst differentially expressed genes, we found increased expression of the c-MYC proto-oncogene (MYC) and its downstream targets in tumor samples. We identified tumors with high and low differential MYC expression and found patients with high-MYC tumors were older and overweight or obese. We also detected elevated expression of the PVT1 long-non-coding RNA (lncRNA) in most tumors and found gains in copy number for both MYC and PVT1 gene loci in 35% of tumors evaluated. Our transcriptome analyses indicate that EOCRC can be sub-classified into groups based on differential MYC expression and suggest that deregulated MYC contributes to CRCs that develop in younger patients.

The Crohn's disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells.

Crohn's disease (CD) is a debilitating inflammatory bowel disease (IBD) that arises from chronic inflammation in the gastrointestinal tract. Genome-wide association studies (GWAS) have identified over 200 single nucleotide polymorphisms (SNPs) that are associated with a predisposition for developing IBD. For the majority, the causal variant and target genes affected are unknown. Here, we investigated the CD-associated SNP rs6651252 that maps to a gene desert region on chromosome 8. We demonstrate that rs6651252 resides within a Wnt responsive DNA enhancer element (WRE) and that the disease associated allele augments binding of the TCF7L2 transcription factor to this region. Using CRISPR/Cas9 directed gene editing and epigenetic modulation, we find that the rs6651252 enhancer regulates expression of the c-MYC proto-oncogene (MYC). Furthermore, we found MYC transcript levels are elevated in patient-derived colonic segments harboring the disease-associated allele in comparison to those containing the ancestral allele. These results suggest that Wnt/MYC signaling contributes to CD pathogenesis and that patients harboring the disease-associated allele may benefit from therapies that target MYC or MYC-regulated genes.

Elevated Colonic Mucin Expression Correlates with Extended Time to Surgery for Ulcerative Colitis Patients.

BACKGROUND AND AIMS: Both genetic and environmental factors contribute to the development and persistence of ulcerative colitis (UC). As supported by differential responses to therapy, multiple subclasses of disease likely comprise UC. We reasoned that profiling the colonic transcriptomes may offer one approach to molecular subtype UC. METHODS: We conducted RNA-sequencing (RNA-seq) on full-thickness colonic tissues from 26 UC patients undergoing colectomy. Hierarchal clustering from transcriptomic data identified disease subsets. Subsets were characterized using differential gene expression analysis, cell type deconvolution, and network analysis. RESULTS: We identified two UC subsets that were distinguished by 957 differentially expressed genes. Cluster 1 was enriched in genes associated with intestinal epithelial cell (IEC) differentiation, while cluster 2 was enriched in genes associated with epithelial-to-mesenchymal transition (EMT) and inflammatory responses. Cluster 1 was associated with an extended time from diagnosis to colectomy [hazard ratio = 0.45 (95% CI: 0.14-0.88); p=0.03]. Of cluster 1 genes, elevated MUC5B, MUC4, and MUC2 expression displayed the strongest correlation with increased time to surgery [hazard ratio = 0.37 (95% CI: 0.11-0.61); p=0.0044]. CONCLUSIONS: Our transcriptome analysis indicates that UC can be sub-classified into at least two molecular signatures. We found that elevated mucin gene expression correlated with prolonged time to colectomy following diagnosis. This work identified MUC5B, MUC4, and MUC2 as potential prognostic indicators of disease severity, as reflected in time to surgery after diagnosis.

Tristetraprolin targets Nos2 expression in the colonic epithelium.

Tristetraprolin (TTP), encoded by the Zfp36 gene, is a zinc-finger protein that regulates RNA stability primarily through association with 3' untranslated regions (3' UTRs) of target mRNAs. While TTP is expressed abundantly in the intestines, its function in intestinal epithelial cells (IECs) is unknown. Here we used a cre-lox system to remove Zfp36 in the mouse epithelium to uncover a role for TTP in IECs and to identify target genes in these cells. While TTP was largely dispensable for establishment and maintenance of the colonic epithelium, we found an expansion of the proliferative zone and an increase in goblet cell numbers in the colon crypts of Zfp36ΔIEC mice. Furthermore, through RNA-sequencing of transcripts isolated from the colons of Zfp36fl/fl and Zfp36ΔIEC mice, we found that expression of inducible nitric oxide synthase (iNos or Nos2) was elevated in TTP-knockout IECs. We demonstrate that TTP interacts with AU-rich elements in the Nos2 3' UTR and suppresses Nos2 expression. In comparison to control Zfp36fl/fl mice, Zfp36ΔIEC mice were less susceptible to dextran sodium sulfate (DSS)-induced acute colitis. Together, these results demonstrate that TTP in IECs targets Nos2 expression and aggravates acute colitis.

The Microbial Ecosystem Distinguishes Chronically Diseased Tissue from Adjacent Tissue in the Sigmoid Colon of Chronic, Recurrent Diverticulitis Patients.

Diverticular disease is commonly associated with the older population in the United States. As individual's age, diverticulae, or herniation of the mucosa through the colonic wall, develop. In 10-25% of individuals, the diverticulae become inflamed, resulting in diverticulitis. The gut ecosystem relies on the interaction of bacteria and fungi to maintain homeostasis. Although bacterial dysbiosis has been implicated in the pathogenesis of diverticulitis, associations between the microbial ecosystem and diverticulitis remain largely unstudied. This study investigated how the cooperative network of bacteria and fungi differ between a diseased area of the sigmoid colon chronically affected by diverticulitis and adjacent non-affected tissue. To identify mucosa-associated microbes, bacterial 16S rRNA and fungal ITS sequencing were performed on chronically diseased sigmoid colon tissue (DT) and adjacent tissue (AT) from the same colonic segment. We found that Pseudomonas and Basidiomycota OTUs were associated with AT while Microbacteriaceae and Ascomycota were enriched in DT. Bipartite co-occurrence networks were constructed for each tissue type. The DT and AT networks were distinct for each tissue type, with no microbial relationships maintained after intersection merge of the groups. Our findings indicate that the microbial ecosystem distinguishes chronically diseased tissue from adjacent tissue.

The MYC 3' Wnt-Responsive Element Drives Oncogenic MYC Expression in Human Colorectal Cancer Cells.

Mutations in components of the Wnt/ß-catenin signaling pathway drive colorectal cancer (CRC) by deregulating expression of downstream target genes including the c-MYC proto-oncogene (MYC). The critical regulatory DNA enhancer elements that control oncogenic MYC expression in CRC have yet to be fully elucidated. In previous reports, we correlated T-cell factor (TCF) and ß-catenin binding to the MYC 3' Wnt responsive DNA element (MYC 3' WRE) with MYC expression in HCT116 cells. Here we used CRISPR/Cas9 to determine whether this element is a critical driver of MYC. We isolated a clonal population of cells that contained a deletion of a single TCF binding element (TBE) within the MYC 3' WRE. This deletion reduced TCF/ß-catenin binding to this regulatory element and decreased MYC expression. Using RNA-Seq analysis, we found altered expression of genes that regulate metabolic processes, many of which are known MYC target genes. We found that 3' WRE-Mut cells displayed a reduced proliferative capacity, diminished clonogenic growth, and a decreased potential to form tumors in vivo. These findings indicate that the MYC 3' WRE is a critical driver of oncogenic MYC expression and suggest that this element may serve as a therapeutic target for CRC.