Two Enhancers Control Transcription of Drosophila muscleblind in the Embryonic Somatic Musculature and in the Central Nervous System

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene.

[1]  M. Swanson,et al.  Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy. , 2009, Human molecular genetics.

[2]  Tyson A. Clark,et al.  Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy , 2010, Nature Structural &Molecular Biology.

[3]  P. Harper,et al.  Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. , 2002, Human molecular genetics.

[4]  Scott Barolo,et al.  New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. , 2004, BioTechniques.

[5]  Marc D. Perry,et al.  ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia , 2012, Genome research.

[6]  M. Ares,et al.  Muscleblind-like 2-Mediated Alternative Splicing in the Developing Brain and Dysregulation in Myotonic Dystrophy , 2012, Neuron.

[7]  A. Masuda,et al.  CUGBP1 and MBNL1 preferentially bind to 3′ UTRs and facilitate mRNA decay , 2012, Scientific Reports.

[8]  Michael P. Eichenlaub,et al.  Combinatorial Binding Leads to Diverse Regulatory Responses: Lmd Is a Tissue-Specific Modulator of Mef2 Activity , 2010, PLoS genetics.

[9]  D. Jacobs,et al.  Deformed autoregulatory element from Drosophila functions in a conserved manner in transgenic mice , 1992, Nature.

[10]  Expression pattern of muscleblind-like proteins differs in differentiating myoblasts. , 2007, Biochemical and biophysical research communications.

[11]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[12]  T. Ayoubi,et al.  Regulation of gene expression by alternative promoters , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  E. Furlong,et al.  Combinatorial binding predicts spatio-temporal cis-regulatory activity , 2009, Nature.

[14]  M. Swanson,et al.  Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. , 2004, Human molecular genetics.

[15]  N. Patel,et al.  Krüppel, a Drosophila segmentation gene, participates in the specification of neurons and glial cells , 1996, Mechanisms of Development.

[16]  C. Sewry,et al.  Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts , 2006, Human Genetics.

[17]  L. Pachter,et al.  rVista for comparative sequence-based discovery of functional transcription factor binding sites. , 2002, Genome research.

[18]  D. Yamamoto,et al.  Genetic Analysis of chaste, a New Mutation of Drosophila melanogaster Characterized by Extremely Low Female Sexual Receptivity , 2009, Journal of neurogenetics.

[19]  M. Swanson,et al.  Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3. , 2003, Gene expression patterns : GEP.

[20]  K. Sobczak,et al.  CAG repeats mimic CUG repeats in the misregulation of alternative splicing , 2011, Nucleic acids research.

[21]  R. Artero,et al.  A conserved motif controls nuclear localization of Drosophila Muscleblind , 2010, Molecules and cells.

[22]  R. Artero,et al.  Alternative splicing regulation by Muscleblind proteins: from development to disease , 2011, Biological reviews of the Cambridge Philosophical Society.

[23]  M. Swanson,et al.  Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. , 2006, Human molecular genetics.

[24]  U. Ohler.  Identification of core promoter modules in Drosophila and their application in accurate transcription start site prediction , 2006, Nucleic acids research.

[25]  Michael G. Poulos,et al.  Muscleblind isoforms are functionally distinct and regulate α-actinin splicing , 2007 .

[26]  S. Tapscott,et al.  RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing* , 2010, The Journal of Biological Chemistry.

[27]  A. Wagers,et al.  Skeletal muscle degenerative diseases and strategies for therapeutic muscle repair. , 2013, Annual review of pathology.

[28]  R. Darnell,et al.  Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice. , 2013, Human molecular genetics.

[29]  D. M. Chenault,et al.  Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR. , 2002, Developmental biology.

[30]  Mary Goldman,et al.  The UCSC Genome Browser database: extensions and updates 2013 , 2012, Nucleic Acids Res..

[31]  Gos Micklem,et al.  Supporting Online Material Materials and Methods Figs. S1 to S50 Tables S1 to S18 References Identification of Functional Elements and Regulatory Circuits by Drosophila Modencode , 2022 .

[32]  D. Dembélé,et al.  Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy , 2011, Nature Structural &Molecular Biology.

[33]  S. Naylor,et al.  Myotonic Dystrophy Type 2 Caused by a CCTG Expansion in Intron 1 of ZNF9 , 2001, Science.

[34]  M. Scott,et al.  Different patterns of transcription from the two Antennapedia promoters during Drosophila embryogenesis. , 1990, Development.

[35]  Robert H. Gross,et al.  SCOPE: a web server for practical de novo motif discovery , 2007, Nucleic Acids Res..

[36]  J. Houseley,et al.  Myotonic dystrophy associated expanded CUG repeat muscleblind positive ribonuclear foci are not toxic to Drosophila. , 2005, Human molecular genetics.

[37]  M. Levine,et al.  Different core promoters possess distinct regulatory activities in the Drosophila embryo. , 1998, Genes & development.

[38]  Drosophila muscleblind Codes for Proteins with One and Two Tandem Zinc Finger Motifs , 2012, PloS one.

[39]  K. Jagla,et al.  Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression. , 2013, Human molecular genetics.

[40]  M. Bellard,et al.  A novel homeobox nkch4 gene from the Drosophila 93E region. , 1993, Gene.

[41]  A. Kania,et al.  P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster. , 1995, Genetics.

[42]  V. Solovyev,et al.  Ab initio gene finding in Drosophila genomic DNA. , 2000, Genome research.

[43]  Eric T. Wang,et al.  Transcriptome-wide Regulation of Pre-mRNA Splicing and mRNA Localization by Muscleblind Proteins , 2012, Cell.

[44]  I. Kiss,et al.  muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. , 1997, Development.

[45]  B. Byrne,et al.  Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy , 2000, The EMBO journal.

[46]  J. Berglund,et al.  RNA binding specificity of Drosophila muscleblind. , 2008, Biochemistry.

[47]  J. Tena,et al.  A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts. , 2005, Genome research.

[48]  Lei Zhou,et al.  Drosophila Muscleblind Is Involved in troponin T Alternative Splicing and Apoptosis , 2008, PloS one.

[49]  J. Urban,et al.  Hunchback is required for the specification of the early sublineage of neuroblast 7-3 in the Drosophila central nervous system. , 2002, Development.

[50]  David E. Housman,et al.  Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member , 1992, Cell.

[51]  Michael P. Eichenlaub,et al.  A temporal map of transcription factor activity: mef2 directly regulates target genes at all stages of muscle development. , 2006, Developmental cell.

[52]  N. Bonini,et al.  Roles of trinucleotide-repeat RNA in neurological disease and degeneration , 2010, Trends in Neurosciences.

[53]  A Prokop,et al.  The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. , 1998, Developmental biology.

[54]  Eric T. Wang,et al.  MBNL proteins repress ES-cell-specific alternative splicing and reprogramming , 2013, Nature.

[55]  C. Rickert,et al.  The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells. , 2004, Developmental biology.

[56]  David J. Arenillas,et al.  JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles , 2009, Nucleic Acids Res..

[57]  Tien Hsu,et al.  RNA-dependent integrin α3 protein localization regulated by the Muscleblind-like protein MLP1 , 2005, Nature Cell Biology.

[58]  RNA toxicity in polyglutamine disorders: concepts, models, and progress of research , 2013, Journal of Molecular Medicine.