Activation of Cryptic Donor Splice Sites within the UDP-Glucuronosyltransferase (UGT)1A First-Exon Region Generates Variant Transcripts That Encode UGT1A Proteins with Truncated Aglycone-Binding Domains.

The human UDP-glucuronosyltransferases (UGTs) have crucial roles in metabolizing and clearing numerous small lipophilic compounds. The UGT1A locus generates nine UGT1A mRNAs, 65 spliced transcripts, and 34 circular RNAs. In this study, our analysis of published UGT-RNA capture sequencing (CaptureSeq) datasets identified novel splice junctions that predict 24 variant UGT1A transcripts derived from ligation of exon 2 to unique sequences within the UGT1A first-exon region using cryptic donor splice sites. Of these variants, seven (1A1_n1, 1A3_n3, 1A4_n4, 1A5_n1, 1A8_n2, 1A9_n2, 1A10_n7) are predicted to encode UGT1A proteins with truncated aglycone-binding domains. We assessed their expression profiles and deregulation in cancer using four RNA sequencing (RNA-Seq) datasets of paired normal and cancerous drug-metabolizing tissues from large patient cohorts. Variants were generally coexpressed with their canonical counterparts with a higher relative abundance in tumor than in normal tissues. Variants showed tissue-specific expression with high interindividual variability but overall low abundance. However, 1A8_n2 showed high abundance in normal and cancerous colorectal tissues, with levels that approached or surpassed canonical 1A8 mRNA levels in many samples. We cloned 1A8_n2 and showed expression of the predicted protein (1A8_i3) in human embryonic kidney (HEK)293T cells. Glucuronidation assays with 4-methylumbelliferone (4MU) showed that 1A8_i3 had no activity and was unable to inhibit the activity of 1A8_i1 protein. In summary, the activation of cryptic donor splice sites within the UGT1A first-exon region expands the UGT1A transcriptome and proteome. The 1A8_n2 cryptic donor splice site is highly active in colorectal tissues, representing an important cis-regulatory element that negatively regulates the function of the UGT1A8 gene through pre-mRNA splicing. SIGNIFICANT STATEMENT: The UGT1A locus generates nine canonical mRNAs, 65 alternately spliced transcripts, and 34 different circular RNAs. The present study reports a series of novel UDP-glucuronosyltransferase (UGT)1A variants resulting from use of cryptic donor splice sites in both normal and cancerous tissues, several of which are predicted to encode variant UGT1A proteins with truncated aglycone-binding domains. Of these, 1A8_n2 shows exceptionally high abundance in colorectal tissues, highlighting its potential role in the first-pass metabolism in gut through the glucuronidation pathway.

A Comprehensive Bioinformatic Analysis of RNA-seq Datasets Reveals a Differential and Variable Expression of Wildtype and Variant UGT1A Transcripts in Human Tissues and Their Deregulation in Cancers.

The UGT1A locus generates over 60 different alternatively spliced transcripts and 30 circular RNAs. To date, v2 and v3 transcripts are the only variant UGT1A transcripts that have been functionally characterized. Both v2 and v3 transcripts encode the same inactive variant UGT1A proteins (i2s) that can negatively regulate glucuronidation activity and influence cancer cell metabolism. However, the abundance and interindividual variability in the expression of v2 and v3 transcripts in human tissues and their potential deregulation in cancers have not been comprehensively assessed. To address this knowledge gap, we quantified the expression levels of v1, v2, and v3 transcripts using RNA-seq datasets with large cohorts of normal tissues and paired normal and tumor tissues from patients with six different cancer types (liver, kidney, colon, stomach, esophagus, and bladder cancer). We found that v2 and v3 abundance varied significantly between different tissue types, and that interindividual variation was also high within the same tissue type. Moreover, the ratio of v2 to v3 variants varied between tissues, implying their differential regulation. Our results showed higher v2 abundance in gastrointestinal tissues than liver and kidney tissues, suggesting a more significant negative regulation of glucuronidation by i2 proteins in gastrointestinal tissues than in liver and kidney tissues. We further showed differential deregulation of wildtype (v1) and variant transcripts (v2, v3) in cancers that generally increased the v2/v1 and/or v3/v1 expression ratios in tumors compared to normal tissues, indicating a more significant role of the variants in tumors. Finally, we report ten novel UGT1A transcripts with novel 3' terminal exons, most of which encode variant proteins with a similar structure to UGT1A_i2 proteins. These findings further emphasize the diversity of the UGT1A transcriptome and proteome.

Drug-drug interactions that alter the exposure of glucuronidated drugs: Scope, UDP-glucuronosyltransferase (UGT) enzyme selectivity, mechanisms (inhibition and induction), and clinical significance.

Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for the pharmaceutical industry. DDIs involving glucuronidated drugs have historically attracted little attention and there is a perception that interactions are of minor clinical relevance. This review critically examines the scope and aetiology of DDIs that result in altered exposure of glucuronidated drugs. Interaction mechanisms, namely inhibition and induction of UDP-glucuronosyltransferase (UGT) enzymes and the potential interplay with drug transporters, are reviewed in detail, as is the clinical significance of known DDIs. Altered victim drug exposure arising from modulation of UGT enzyme activities is relatively common and, notably, the incidence and importance of UGT induction as a DDI mechanism is greater than generally believed. Numerous DDIs are clinically relevant, resulting in either loss of efficacy or an increased risk of adverse effects, necessitating dose individualisation. Several generalisations relating to the likelihood of DDIs can be drawn from the known substrate and inhibitor selectivities of UGT enzymes, highlighting the importance of comprehensive reaction phenotyping studies at an early stage of drug development. Further, rigorous assessment of the DDI liability of new chemical entities that undergo glucuronidation to a significant extent has been recommended recently by regulatory guidance. Although evidence-based approaches exist for the in vitro characterisation of UGT enzyme inhibition and induction, the availability of drugs considered appropriate for use as 'probe' substrates in clinical DDI studies is limited and this should be a research priority.

The Somatic Mutation Landscape of UDP-Glycosyltransferase (UGT) Genes in Human Cancers.

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5' UTR, 3' UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes.

Regulation of human UDP-glycosyltransferase (UGT) genes by miRNAs.

The human UGT gene superfamily is divided into four subfamilies (UGT1, UGT2, UGT3 and UGT8) that encodes 22 functional enzymes. UGTs are critical for the metabolism and clearance of numerous endogenous and exogenous compounds, including steroid hormones, bile acids, bilirubin, fatty acids, carcinogens, and therapeutic drugs. Therefore, the expression and activities of UGTs are tightly regulated by multiple processes at the transcriptional, post-transcriptional and post-translational levels. During recent years, nearly twenty studies have investigated the post-transcriptional regulation of UGT genes by miRNAs using human cancer cell lines (predominantly liver cancer). Overall, 14 of the 22 UGT mRNAs (1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B10, 2B15, 2B17, UGT8) have been shown to be regulated by various miRNAs through binding to their respective 3' untranslated regions (3'UTRs). Three 3'UTRs (UGT1A, UGT2B7 and UGT2B15) contain the largest number of functional miRNA target sites; in particular, the UGT1A 3'UTR contains binding sites for 12 miRNAs (548d-5p, 183-5p, 214-5p, 486-3p, 200a-3p, 491-3p, 141-3p, 298, 103b, 376b-3p, 21-3p, 1286). Although all nine UGT1A family members have the same 3'UTR, these miRNA target sites appear to be functional in an isoform-specific and cellular context-dependent manner. Collectively, these observations demonstrate that miRNAs represent important post-transcriptional regulators of the UGT gene superfamily. In this article, we present a comprehensive review of reported UGT/miRNA regulation studies, describe polymorphisms within functional miRNA target sites that may affect their functionalities, and discuss potential cooperative and competitive regulation of UGT mRNAs by miRNAs through adjacently located miRNA target sites.

The Expression Profiles and Deregulation of UDP-Glycosyltransferase (UGT) Genes in Human Cancers and Their Association with Clinical Outcomes.

The human UDP-glycosyltransferase (UGTs) superfamily has 22 functional enzymes that play a critical role in the metabolism of small lipophilic compounds, including carcinogens, drugs, steroids, lipids, fatty acids, and bile acids. The expression profiles of UGT genes in human cancers and their impact on cancer patient survival remains to be systematically investigated. In the present study, a comprehensive analysis of the RNAseq and clinical datasets of 9514 patients from 33 different TCGA (the Genome Cancer Atlas) cancers demonstrated cancer-specific UGT expression profiles with high interindividual variability among and within individual cancers. Notably, cancers derived from drug metabolizing tissues (liver, kidney, gut, pancreas) expressed the largest number of UGT genes (COAD, KIRC, KIRP, LIHC, PAAD); six UGT genes (1A6, 1A9, 1A10, 2A3, 2B7, UGT8) showed high expression in five or more different cancers. Kaplan-Meier plots and logrank tests revealed that six UGT genes were significantly associated with increased overall survival (OS) rates [UGT1A1 (LUSC), UGT1A6 (ACC), UGT1A7 (ACC), UGT2A3 (KIRC), UGT2B15 (BLCA, SKCM)] or decreased OS rates [UGT2B15 (LGG), UGT8 (UVM)] in specific cancers. Finally, differential expression analysis of 611 patients from 12 TCGA cancers identified 16 UGT genes (1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A3, 2B4, 2B7, 2B11, 2B15, 3A1, 3A2, UGT8) that were up/downregulated in at least one cancer relative to normal tissues. In conclusion, our data show widespread expression of UGT genes in cancers, highlighting the capacity for intratumoural drug metabolism through the UGT conjugation pathway. The data also suggests the potentials for specific UGT genes to serve as prognostic biomarkers or therapeutic targets in cancers.

Circular RNAs of UDP-Glycosyltransferase (UGT) Genes Expand the Complexity and Diversity of the UGT Transcriptome.

The human UDP-glycosyltransferase (UGT) gene superfamily generates 22 canonical transcripts coding for functional enzymes and also produces nearly 150 variant UGT transcripts through alternative splicing and intergenic splicing. In the present study, our analysis of circRNA databases identified backsplicing events that predicted 85 circRNAs from UGT genes, with 33, 11, and 19 circRNAs from UGT1A, UGT2B4, UGT8, respectively. Most of these UGT circRNAs were reported by one database and had low abundance in cell- or tissue-specific contexts. Using reverse-transcriptase polymerase chain reaction with divergent primers and cDNA samples from human tissues and cell lines, we found 13 circRNAs from four UGT genes: UGT1A (three), UGT2B7 (one), UGT2B10 (one), and UGT8 (eight). Notably, all eight UGT8 circRNAs contain open reading frames that include the canonical start AUG codon and encode variant proteins that all have the common 274-amino acidN-terminal region of wild-type UGT8 protein. We further showed that one UGT8 circRNA (circ_UGT8-1) was broadly expressed in human tissues and cell lines, resistant to RNase R digestion, and predominately present in the cytoplasm. We cloned five UGT8 circRNAs into the Zinc finger with KRAB and SCAN domains 1 vector and transfected them into HEK293T cells. All these vectors produced both circRNAsand linear transcripts with varying circular/linear ratios (0.17-1.14).Western blotting and mass spectrometry assays revealed that only linear transcripts and not circRNAs were translated. In conclusion, our findings of nearly 100 circRNAs greatly expand the complexity and diversity of the UGT transcriptome; however, UGT circRNAs are expressed at a very low level in specific cellular contexts, and their biologic functions remain to be determined. SIGNIFICANCE STATEMENT: The human UGT gene transcriptome comprises 22 canonical transcripts coding for functional enzymes and approximately 150 alternatively spliced and chimeric variant transcripts. The present study identified nearly 100 circRNAs from UGT genes, thus greatly expanding the complexity and diversity of the UGT transcriptome. UGT circRNAs were expressed broadly in human tissues and cell lines; however, most showed very low abundance in tissue- and cell-specific contexts, and therefore their biological functions remain to be investigated.

ß-Catenin is essential for differentiation of primary myoblasts via cooperation with MyoD and α-catenin.

Canonical Wnts promote myoblast differentiation; however, the role of ß-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated ß-catenin-null primary adult mouse myoblasts, we found that ß-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a ß-catenin-TCF/LEF interaction is not required for differentiation, and that ß-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by ß-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of ß-catenin with α-catenin was important for efficient differentiation. Overall the study suggests dual roles for ß-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an α-catenin-dependent membrane function that helps control cell-cell interactions. ß-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of ß-catenin and thus prevent precocious/excessive myoblast fusion.

Small molecule inhibition of DDAH1 significantly attenuates triple negative breast cancer cell vasculogenic mimicry in vitro.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a key enzyme involved in the metabolism of the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA). Increased DDAH1 expression and subsequent increased NO production have been recently linked to cancer. Specifically, DDAH1 is implicated in establishment of a vascular network by tumour cells, vasculogenic mimicry (VM), which is strongly associated with tumour progression and poor patient prognosis. The use of DDAH1 inhibitors as potential therapeutic agents thus represents a growing field of interest. Here we describe a UPLC-MS assay to quantify stability and intracellular concentration of two small molecule DDAH1 inhibitors synthesised by our group, ZST316 and ZST152, following incubation with MDA-MB-231 breast cancer cells. In an in vitro assay of VM, both DDAH1 inhibitors significantly attenuated formation of capillary-like tube structures in a dose-dependent fashion. This was not due to cell toxicity or altered cell proliferation, but may be due in part to inhibition of cell migration. Mechanistically, we demonstrate significant modulation of the endogenous DDAH/ADMA/NO pathway following exposure of 100 µM ZST316 or ZST152: a 40% increase in the DDAH1 substrate ADMA, and a 38% decrease in the DDAH1 product l-citrulline. This study represents the first evidence for therapeutic inhibition of DDAH1 by small molecules in breast cancer.

Inhibition of Dimethylarginine Dimethylaminohydrolase (DDAH) Enzymes as an Emerging Therapeutic Strategy to Target Angiogenesis and Vasculogenic Mimicry in Cancer.

The small free radical gas nitric oxide (NO) plays a key role in various physiological and pathological processes through enhancement of endothelial cell survival and proliferation. In particular, NO has emerged as a molecule of interest in carcinogenesis and tumor progression due to its crucial role in various cancer-related events including cell invasion, metastasis, and angiogenesis. The dimethylarginine dimethylaminohydrolase (DDAH) family of enzymes metabolize the endogenous nitric oxide synthase (NOS) inhibitors, asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), and are thus key for maintaining homeostatic control of NO. Dysregulation of the DDAH/ADMA/NO pathway resulting in increased local NO availability often promotes tumor growth, angiogenesis, and vasculogenic mimicry. Recent literature has demonstrated increased DDAH expression in tumors of different origins and has also suggested a potential ADMA-independent role for DDAH enzymes in addition to their well-studied ADMA-mediated influence on NO. Inhibition of DDAH expression and/or activity in cell culture models and in vivo studies has indicated the potential therapeutic benefit of this pathway through inhibition of both angiogenesis and vasculogenic mimicry, and strategies for manipulating DDAH function in cancer are currently being actively pursued by several research groups. This review will thus provide a timely discussion on the expression, regulation, and function of DDAH enzymes in regard to angiogenesis and vasculogenic mimicry, and will offer insight into the therapeutic potential of DDAH inhibition in cancer based on preclinical studies.

Intergenic Splicing between Four Adjacent UGT Genes (2B15, 2B29P2, 2B17, 2B29P1) Gives Rise to Variant UGT Proteins That Inhibit Glucuronidation via Protein-Protein Interactions.

Recent studies have investigated alternative splicing profiles of UDP-glucuronosyltransferase (UGT) genes and identified over 130 different alternatively spliced UGT transcripts. Although UGT genes are highly clustered, the formation of chimeric transcripts by intergenic splicing between two or more UGT genes has not yet been reported. This study identified 12 chimeric transcripts (chimeras A-L) containing exons from two or three genes of the four neighboring UGT genes (UGT2B15, UGT2B29P2, UGT2B17, and UGT2B29P1) in human liver and prostate cancer cells. These chimeras typically contain the first five exons of UGT2B15 or UGT2B17 (exons 1-5) spliced to a terminal exon (exon 6) from a downstream UGT gene. Hence they encode truncated UGTs with novel C-terminal peptides. Functional assays of representative chimeric UGT proteins (termed chimeric UGT2B15 and chimeric UGT2B17) showed that they are inactive and can repress the activity of wild-type UGTs. Coimmunoprecipitation assays demonstrated heterotypic interactions between chimeric UGT2B15 (or chimeric UGT2B17) and the UGT2B7 protein. Thus oligomerization of the chimeric UGTs with wild-type UGTs may explain their inhibitory activity. Studies in breast and prostate cancer cells showed that both wild-type and chimeric UGT2B15 and UGT2B17 transcripts are regulated in a similar way at the transcriptional level by sex hormones through their canonical promoters but are differentially regulated at the post-transcriptional level by micro-RNA 376c via their unique 3'-untranslated regions. In conclusion, the formation of chimeric transcripts by intergenic splicing among UGT genes represents a novel mechanism contributing to the diversity of the human UGT transcriptome and proteome. The differential post-transcriptional regulation of wild-type and variant transcripts by micro-RNAs may contribute to their deregulated expression in cancer.

Cooperative Regulation of Intestinal UDP-Glucuronosyltransferases 1A8, -1A9, and 1A10 by CDX2 and HNF4α Is Mediated by a Novel Composite Regulatory Element.

The gastrointestinal tract expresses several UDP-glucuronosyltransferases (UGTs) that act as a first line of defense against dietary toxins and contribute to the metabolism of orally administered drugs. The expression of UGT1A8, UGT1A9, and UGT1A10 in gastrointestinal tissues is known to be at least partly directed by the caudal homeodomain transcription factor, CDX2. We sought to further define the factors involved in regulation of the UGT1A8-1A10 genes and identified a novel composite element located within the proximal promoters of these three genes that binds to both CDX2 and the hepatocyte nuclear factor (HNF) 4α, and mediates synergistic activation by these factors. We also show that HNF4α and CDX2 are required for the expression of these UGT genes in colon cancer cell lines, and show robust correlation of UGT expression with CDX2 and HNF4α levels in normal human colon. Finally, we show that these factors are involved in the differential expression pattern of UGT1A8 and UGT1A10, which are intestinal specific, and that of UGT1A9, which is expressed in both intestine and liver. These studies lead to a model for the developmental patterning of UGT1A8, UGT1A9, and UGT1A10 in hepatic and/or extrahepatic tissues involving discrete regulatory modules that may function (independently and cooperatively) in a context-dependent manner.

MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1- MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

Barx2 and Pax7 Regulate Axin2 Expression in Myoblasts by Interaction with ß-Catenin and Chromatin Remodelling.

Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T-cell factor/lymphoid enhancer factor (TCF/LEF) and ß-catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/ß-catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits ß-catenin and the coactivator GRIP-1, and induces local H3K-acetylation. Barx2 also promotes nuclear localization of ß-catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2-mediated H3K-acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation-competent and -incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/ß-catenin complex. Stem Cells 2016;34:2169-2182.

Barx2 and Pax7 have antagonistic functions in regulation of wnt signaling and satellite cell differentiation.

The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle-specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors ß-catenin and T cell-factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of ß-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds ß-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation.

Barx2 is expressed in satellite cells and is required for normal muscle growth and regeneration.

Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2(-/-) mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2(+/-) mice. Barx2(-/-) myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation.

The novel UDP glycosyltransferase 3A2: cloning, catalytic properties, and tissue distribution.

The human UDP glycosyltransferase (UGT) 3A family is one of three families involved in the metabolism of small lipophilic compounds. Members of these families catalyze the addition of sugar residues to chemicals, which enhances their excretion from the body. The UGT1 and UGT2 family members primarily use UDP glucuronic acid to glucuronidate numerous compounds, such as steroids, bile acids, and therapeutic drugs. We showed recently that UGT3A1, the first member of the UGT3 family to be characterized, is unusual in using UDP N-acetylglucosamine as sugar donor, rather than UDP glucuronic acid or other UDP sugar nucleotides (J Biol Chem 283:36205-36210, 2008). Here, we report the cloning, expression, and characterization of UGT3A2, the second member of the UGT3 family. Like UGT3A1, UGT3A2 is inactive with UDP glucuronic acid as sugar donor. However, in contrast to UGT3A1, UGT3A2 uses both UDP glucose and UDP xylose but not UDP N-acetylglucosamine to glycosidate a broad range of substrates including 4-methylumbelliferone, 1-hydroxypyrene, bioflavones, and estrogens. It has low activity toward bile acids and androgens. UGT3A2 transcripts are found in the thymus, testis, and kidney but are barely detectable in the liver and gastrointestinal tract. The low expression of UGT3A2 in the latter, which are the main organs of drug metabolism, suggests that UGT3A2 has a more selective role in protecting the organs in which it is expressed against toxic insult rather than a more generalized role in drug metabolism. The broad substrate and novel UDP sugar specificity of UGT3A2 would be advantageous for such a function.

The homeobox transcription factor Barx2 regulates plasticity of young primary myofibers.

BACKGROUND: Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool. METHODOLOGY/PRINCIPAL FINDINGS: We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2. CONCLUSIONS/SIGNIFICANCE: We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation.