Expression In Non-muscle Cells Research Articles

ACTB::GLI1 Gene Fusion Small Bowel Mesenteric Tumor; a Case Report and Review of the Literature

Abstract Introduction/Objective GLI1 encodes a transcription factor that targets cell cycle regulators affecting stem cell proliferation. Activation of the GLI (glioma-associated oncogene homologue 1). GLI activity in adult tissues is restricted but has been identified in various neoplasms, as a result of mutations in the PTCH (patched) or SMOH (smoothened) genes, encoding components of the sonic hedgehog pathway, or by amplification of GLI. The B-actin (ACTB) gene, encoding an essential cytoskeletal component, is under the control of a conserved, strong, and complex promoter that assures a high level of expression in non-muscle cells. ACTB::GLI1 gene fusions have been reported in the tongue as the pathognomonic genetic abnormality defining an unusual subset of actin-positive, perivascular tumors with t (7;12) translocation. Methods/Case Report We report a case of 53-year-old male presented to the hospital with dizziness and bloody stools. CT scan of the abdomen and pelvis revealed a 10.5 x 7.7 cm large complex cystic and solid mass of the right lower quadrant of the abdomen adjacent to the cecum and terminal ileum. The patient has a past medical history of testicular germ cell tumor and underwent orchiectomy with adjuvant chemotherapy 30 years ago. The mass was excised and histological examination of the mass showed an unencapsulated, relatively circumscribed neoplasm composed of sheets of monomorphic bland spindle to ovoid cells. Immunohistochemical (IHC) stains, including pan- CK, SALL4, c-Kit, CD34, SMA, MDM2, HMB45, and MART1, were all negative. NGS sarcoma fusion panel was done and revealed ACTB::GLI1 gene fusion. The tumor was finally diagnosed as GLI1 -rearranged enteric tumor. Results (if a Case Study enter NA) NA Conclusion our case, in addition to, previously reported cases in the literature support the existence of a newly identified, seemingly discrete group of soft tissue tumors at different body locations. The morphology of these tumors is somewhat variable, making identifying and diagnosing these tumors more challenging. In addition to the morphologic variance, the diagnosis of these tumors in unusual locations is not uncommon, with some variations in IHC staining patterns. Identifying the molecular pattern of these tumors and the biological mechanism will facilitate more effective treatment options for patients, including targeted therapy.

Tissue-specific expression of myosin phosphatase subunits and isoforms in smooth muscle of mice and humans.

Alternative splicing of exon24 (E24) of myosin phosphatase targeting subunit 1 (Mypt1) by setting sensitivity to nitric oxide (NO)/cGMP-mediated relaxation is a key determinant of smooth muscle function. Here we defined expression of myosin phosphatase (MP) subunits and isoforms by creation of new genetic mouse models, assay of human and mouse tissues, and query of public databases. A Mypt1-LacZ reporter mouse revealed that Mypt1 transcription is turned on early in development during smooth muscle differentiation. Mypt1 is not as tightly restricted in its expression as smooth muscle myosin heavy chain (Myh11) and its E6 splice variant. Mypt1 is enriched in mature smooth versus nonmuscle cells. The E24 splice variant and leucine zipper minus protein isoform that it encodes is enriched in phasic versus tonic smooth muscle. In the vascular system, E24 splicing increases as vessel size decreases. In the gastrointestinal system, E24 splicing is most predominant in smooth muscle of the small intestine. Tissue-specific expression of MP subunits and Mypt1 E24 splicing is conserved in humans, whereas a splice variant of the inhibitory subunit (CPI-17) is unique to humans. A Mypt1 E24 mini-gene splicing reporter mouse generated to define patterns of E24 splicing in smooth muscle cells (SMCs) dispersed throughout the organ systems was unsuccessful. In summary, expression of Mypt1 and splicing of E24 is part of the program of smooth muscle differentiation, is further enhanced in phasic smooth muscle, and is conserved in humans. Its low-level expression in nonmuscle cells may confound its measurement in tissue samples.

721. Long-Term (2-8 years) Body-Wide Expression of AAV9-Minidystrophin Gene in Golden Retriever Muscular Dystrophy Dogs After Regional Limb Vein Injection

Previously we have shown that AAV9-minidystrophin gene delivery by isolated hindlimb vein injection with pressure was able to obtain widespread gene expression, not only in injected limb but also in muscles body-wide. This phenomenon was attributed to both leaky tourniquet blockade and/or AAV9 vector retained in the vasculature space that later entered systemic circulation. Here we report the results of long-term study up to 8 years. Methods: Five GRMD dogs were treated with the same vector dose of 1 × 1013 v.g./kg. Dog Jelly (2.5 months old; 6.3 kg) was given AAV9-CMV-opti-hMinidys (codon-optimized human minidystrophin gene); Dogs Jasper and Peridot (4 mon old; 12.2 kg & 12.5 kg) were given AAV9-CMV-cMinidys (canine minidystrophin gene). Dogs Rutela and Laredo (14 mon old; 15.5 kg & 15.3 kg) were given AAV-CK-opti-cMinidys. No immuno-suppressant was used. The vectors were injected via the great saphenous with a tourniquet positioned at the proximal pelvic extremity (first 3 dogs) or above the knee (last 2 dogs) to block the blood circulation for a total of 10 minutes. MRI imaging after vector injection in Jelly confirmed vector fluid and muscle enlargement in the injected limb. Muscle biopsy and final necropsy were performed at various time points (from 2 months to 8 years) and analyzed for gene expression and immune responses. Results: Immunofluorescent (IF) staining showed that all 5 dogs obtained long-term minidys expression in a majority of muscle samples examined up to final necropsy. Contractile force measurement showed partial improvement when compared to the untreated dogs. While the percentages of minidys positive myofibers varied among different muscles, certain muscles had greater than 90% of myofibers positive upon necropsy. Importantly, the human minidys expression persisted for 8 years in Jelly despite initial inflammation in injected limb. Overall gene expression was largely stable. For example, positive myofibers in cranial sartorius muscle remained comparable throughout the 5 time points, from 2, 7 months to 1, 4 and 8 years. Minidys was also observed in approximately 20% of the cardiomyocytes. Interestingly, injected limb had lower expression than non-injected limb, suggesting procedure related inflammation and partial CMV promoter shutdown. Jelly remained ambulant throughout the more than 8-year post treatment study and was euthanized due to cardiomyopathy in the final year. DNA sequencing showed that Jelly did not carry the recently reported disease-modifying Jagged 1 mutation found in two phenotypically mild GRMD dogs. However, it is not possible to attribute Jelly's long-term survival to gene therapy because in the past a few GRMD dogs in the same colony survived to 5 years without treatment. For the other 4 dogs who were larger at the age of treatment, the injected limb consistently showed higher expression and no procedure related inflammation throughout the time course of 2 years. Moreover, no tumors were found in any of the treated dogs. Final analyses of necropsy samples on vector distribution and Western blot are underway to evaluate the efficiency of gene transfer and expression. Conclusions: 1) AAV9 can render long-term and significant systemic minidys gene expression without immune suppression upon isolated limb vein injection. 2) The human minidys gene and pressurized perfusion might have caused inflammation and innate immune responses in the injected limb in Jelly. 3) Muscle-specific promoter is preferable for reducing expression in non-muscle cells. 4) These findings support the feasibility of AAV9-CK-minidys gene therapy in human DMD patients.

Triadin regulation of the ryanodine receptor complex.

The calcium release complex is the major player in excitation-contraction coupling, both in cardiac and skeletal muscle. The core of the complex is the ryanodine receptor, and triadin is a regulating protein. Nevertheless, the precise function of triadin is only partially understood. Besides its function in the anchoring of calsequestrin at the triad/dyad, our recent results allow us to propose hypotheses on new triadin scaffolding functions, based on the studies performed using different models, from triadin knockout mice to human patients, and expression in non-muscle cells, taking into account the presence of multiple triadin isoforms.

Mapping and functional characterization of the murine Smoothelin-like 1 promoter

BackgroundSmoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.ResultsWe mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence.ConclusionSmtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

Myocardin Marks the Earliest Cardiac Gene Expression and Plays an Important Role in Heart Development

Myocardin belongs to the SAP domain family of transcription factors and is expressed specifically in cardiac and smooth muscle during embryogenesis and in adulthood. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. However, the in vivo function of myocardin during cardiogenesis is not completely understood. Here we clone myocardin from chick embryonic hearts and show that myocardin protein sequences are highly conserved cross species. Detailed studies of chick myocardin expression reveal that myocardin is expressed in cardiac and smooth muscle lineage during early embryogenesis, similar to that found in mouse. Interestingly, the expression of myocardin in the heart was found enriched in the outflow tract and the sinoatrial segments shortly after the formation of linear heart tube. Such expression pattern is also maintained in later developing embryos, suggesting that myocardin may play a unique role in the formation of those cardiac modules. Similar to its mouse counterpart, chick myocardin is able to activate cardiac and smooth muscle promoter reporter genes and induce smooth muscle gene expression in nonmuscle cells. Ectopic overexpression of myocardin enlarged the embryonic chick heart. Conversely, repression of the endogenous chick myocardin using antisense oligonucleotides or a dominant negative mutant form of myocardin inhibited cardiogenesis. Together, our data place myocardin as one of the earliest cardiac marker genes for cardiogenesis and support the idea that myocardin plays an essential role in cardiac gene expression and cardiogenesis.

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes.

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and alpha-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and alpha-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.

Single-stranded DNA-binding Proteins PURα and PURβ Bind to a Purine-rich Negative Regulatory Element of the α-Myosin Heavy Chain Gene and Control Transcriptional and Translational Regulation of the Gene Expression: IMPLICATIONS IN THE REPRESSION OF α-MYOSIN HEAVY CHAIN DURING HEART FAILURE

The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells.

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Flanking Sequences Modulate the Cell Specificity of M-CAT Elements

M-CAT elements mediate both muscle-specific and non-muscle-specific transcription. We used artificial promoters to dissect M-CAT elements derived from the cardiac troponin T promoter, whose regulation is highly striated muscle specific. We show that muscle-specific M-CAT-dependent expression requires two distinct components: the core heptameric M-CAT motif (5'-CATTCCT-3'), which constitutes the canonical binding site for TEF-1-related proteins, and specific sequences immediately flanking the core motif that bind an additional factor(s). These factors are found in higher-order M-CAT DNA-protein complexes with TEF-1 proteins. Non-muscle-specific promoters are produced when the sequences flanking the M-CAT motif are removed or modified to match those of non-muscle-specific promoters such as the simian virus 40 promoter. Moreover, a mutation of the 5'-flanking region of the cardiac troponin T M-CAT-1 element upregulated expression in nonmuscle cells. That mutation also disrupts a potential E box that apparently does not bind myogenic basic helix-loop-helix proteins. We propose a model in which M-CAT motifs are potentially active in many cell types but are modulated through protein binding to specific flanking sequences. In nonmuscle cells, these flanking sequences bind a factor(s) that represses M-CAT-dependent activity. In muscle cells, on the other hand, the factor(s) binding to these flanking sequences contributes to both the cell specificity and the overall transcriptional strength of M-CAT-dependent promoters.

Serum-inducible factors binding to an activating transcription factor motif regulate transcription of the Id2A promoter during myogenic differentiation.

Expression of Id, a dominant negative regulator of helix-loop-helix transcription factors, is tightly regulated during cellular differentiation. In this study, we have defined the sequences responsible for the transcriptional regulation of the human Id2A gene. 5' deletion and site-specific mutation analyses of the Id2A promoter showed that both an Sp-1 site and an activating transcription factor (ATF)-like site (referred to as IdATF sites) are required for expression in C2 cells, whereas these sites are not essential for the expression in nonmuscle cells, including HeLa and 10T1/2 cells. Gel shift assays revealed nuclear factors with specific binding to IdATF sites in both C2 and HeLa cells, which are efficiently competed by either legitimate ATF- or AP-1 binding sites. DNA binding activity to IdATF sites was clearly decreased during C2 cell differentiation and rapidly reactivated by treatment with either phorbol 12-myristate 13-acetate or serum, which correlated with the induction of endogenous Id2A mRNA expression. In addition, we show that overexpression of the catalytic domain of protein kinase C leads to the activation of the Id2A promoter through IdATF sites in C2 cells. These results suggest a model in which down-modulation of IdATF site binding activity by mitogen depletion contributes to the down-regulation of Id2A gene expression that is essential for myogenic differentiation.

A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

DNA-binding site for two skeletal actin promoter factors is important for expression in muscle cells.

Two nuclear factors bind to the same site in the chicken skeletal actin promoter. Mutations in the footprint sequence which eliminate detectable binding decrease expression in transfected skeletal muscle cells by a factor of 25 to 50 and do not elevate the low expression in nonmuscle cells. These results show that the factor-binding site contributes to the activation of expression in muscle cells and that it alone does not contribute significantly to repress expression in nonmuscle cells.

DNA-binding site for two skeletal actin promoter factors is important for expression in muscle cells.

Two nuclear factors bind to the same site in the chicken skeletal actin promoter. Mutations in the footprint sequence which eliminate detectable binding decrease expression in transfected skeletal muscle cells by a factor of 25 to 50 and do not elevate the low expression in nonmuscle cells. These results show that the factor-binding site contributes to the activation of expression in muscle cells and that it alone does not contribute significantly to repress expression in nonmuscle cells.

Reprogramming cell differentiation in the absence of DNA synthesis

We examined whether the activation of muscle gene expression in nonmuscle cells required DNA synthesis. Human fibroblasts from amniotic fluid and fetal lung were fused with differentiated mouse muscle cells in the presence or absence of the DNA synthesis inhibitor, cytosine arabinoside. In the stable heterokaryons formed, the human contractile enzyme, MM-creatine kinase (CK), and the cell surface antigen, 5.1H11, were detected in comparable amounts regardless of whether DNA synthesis had occurred. A single cell analysis revealed that the efficiency of gene activation was high and that DNA synthetic activity was not affected by the ratio of muscle to nonmuscle nuclei in the heterokaryons. In addition, muscle gene expression was not restricted to the G1 phase of the cell cycle. We conclude that cell differentiation can be reprogrammed in heterokaryons regardless of cell cycle phase and in the absence of detectable DNA synthesis.