Lmx1b-targeted cis-regulatory modules involved in limb dorsalization

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (n=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis-regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization. Summary: Correlating Lmx1b-binding sites with Lmx1b-regulated genes during mouse limb development uncovers cis-regulatory modules and their gene targets involved in limb dorsal-ventral identity.

[1]  Martin Vingron,et al.  Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding , 2017, Genome research.

[2]  A. Visel,et al.  Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13 , 2016, Cell reports.

[3]  A. Reddi,et al.  Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton , 2016, PLoS genetics.

[4]  William Stafford Noble,et al.  The MEME Suite , 2015, Nucleic Acids Res..

[5]  F. Escande,et al.  Nail–Patella Syndrome: clinical and molecular data in 55 families raising the hypothesis of a genetic heterogeneity , 2015, European Journal of Human Genetics.

[6]  William Stafford Noble,et al.  Motif-based analysis of large nucleotide data sets using MEME-ChIP , 2014, Nature Protocols.

[7]  M. Tessier-Lavigne,et al.  Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor , 2014, PloS one.

[8]  Denis Paquette,et al.  Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of HoxA Genes in Developing Limbs , 2013, PLoS genetics.

[9]  D. Duboule,et al.  Topology of mammalian developmental enhancers and their regulatory landscapes , 2013, Nature.

[10]  R. Nowak,et al.  Extracellular Matrix Collagen Alters Cell Proliferation and Cell Cycle Progression of Human Uterine Leiomyoma Smooth Muscle Cells , 2013, PloS one.

[11]  Laura E. DeMare,et al.  The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb , 2013, Cell.

[12]  L. Reynard,et al.  The Identification of Trans-acting Factors That Regulate the Expression of GDF5 via the Osteoarthritis Susceptibility SNP rs143383 , 2013, PLoS genetics.

[13]  Laura E. DeMare,et al.  The genomic landscape of cohesin-associated chromatin interactions , 2013, Genome research.

[14]  F. Spitz,et al.  An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. , 2013, Developmental cell.

[15]  Kozo Nakamura,et al.  SOX11 contributes to the regulation of GDF5 in joint maintenance , 2013, BMC Developmental Biology.

[16]  James Taylor,et al.  Genomic approaches towards finding cis-regulatory modules in animals , 2012, Nature Reviews Genetics.

[17]  K. Kanaya,et al.  Detection of genes regulated by Lmx1b during limb dorsalization , 2012, Development, growth & differentiation.

[18]  Jesse R. Dixon,et al.  Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions , 2012, Nature.

[19]  Timothy J. Durham,et al.  Combinatorial Patterning of Chromatin Regulators Uncovered by Genome-wide Location Analysis in Human Cells , 2011, Cell.

[20]  Ralph Weissleder,et al.  WNT5A/JNK and FGF/MAPK Pathways Regulate the Cellular Events Shaping the Vertebrate Limb Bud , 2010, Current Biology.

[21]  A. Kania,et al.  Identification of genes controlled by LMX1B in E13.5 mouse limbs , 2010, Developmental dynamics : an official publication of the American Association of Anatomists.

[22]  David A. Orlando,et al.  Mediator and Cohesin Connect Gene Expression and Chromatin Architecture , 2010, Nature.

[23]  Cory Y. McLean,et al.  GREAT improves functional interpretation of cis-regulatory regions , 2010, Nature Biotechnology.

[24]  D. DeSimone,et al.  The extracellular matrix in development and morphogenesis: a dynamic view. , 2010, Developmental biology.

[25]  Kristian Helin,et al.  Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes , 2010, Nucleic acids research.

[26]  Stefan Mundlos,et al.  Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation. , 2009, Gene expression patterns : GEP.

[27]  Joseph C. Pearson,et al.  Transcriptional autoregulation in development , 2009, Current Biology.

[28]  A. Munnich,et al.  Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence , 2009, Nature Genetics.

[29]  Nathaniel D. Heintzman,et al.  Histone modifications at human enhancers reflect global cell-type-specific gene expression , 2009, Nature.

[30]  A. Visel,et al.  ChIP-seq accurately predicts tissue-specific activity of enhancers , 2009, Nature.

[31]  K. Kanaya,et al.  IDENTIFICATION OF DEVELOPMENTAL ENHANCERS USING TARGETED REGIONAL ELECTROPORATION (TREP) OF EVOLUTIONARILY CONSERVED REGIONS , 2008 .

[32]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

[33]  A. Kania,et al.  Identification of genes controlled by LMX1B in the developing mouse limb bud , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[34]  James Sharpe,et al.  Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme , 2007, Development.

[35]  Randy L. Johnson,et al.  The podocyte-specific inactivation of Lmx1b, Ldb1 and E2a yields new insight into a transcriptional network in podocytes. , 2007, Developmental biology.

[36]  Yusuke Nakamura,et al.  A functional polymorphism in the 5′ UTR of GDF5 is associated with susceptibility to osteoarthritis , 2007, Nature Genetics.

[37]  Inna Dubchak,et al.  VISTA Enhancer Browser—a database of tissue-specific human enhancers , 2006, Nucleic Acids Res..

[38]  D. Duboule,et al.  A mouse model for human short-stature syndromes identifies Shox2 as an upstream regulator of Runx2 during long-bone development. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Soares,et al.  Pax9 and Jagged1 act downstream of Gli3 in vertebrate limb development , 2005, Mechanisms of Development.

[40]  D. Duboule,et al.  Comparative analysis of genes downstream of the Hoxd cluster in developing digits and external genitalia , 2005, Development.

[41]  S. Sereika,et al.  Skeletal integrity in patients with nail patella syndrome. , 2005, The Journal of clinical endocrinology and metabolism.

[42]  Lior Pachter,et al.  VISTA: computational tools for comparative genomics , 2004, Nucleic Acids Res..

[43]  J. Gulcher,et al.  Genomewide scan for hand osteoarthritis: a novel mutation in matrilin-3. , 2003, American journal of human genetics.

[44]  H. Kondoh,et al.  Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. , 2003, Developmental cell.

[45]  A. Green,et al.  Nail patella syndrome: a review of the phenotype aided by developmental biology , 2003, Journal of medical genetics.

[46]  D. Kingsley,et al.  Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes. , 2003, Developmental biology.

[47]  Cameron S. Osborne,et al.  Long-range chromatin regulatory interactions in vivo , 2002, Nature Genetics.

[48]  M. Smidt,et al.  CNS Expression Pattern of Lmx1b and Coexpression with Ptx Genes Suggest Functional Cooperativity in the Development of Forebrain Motor Control Systems , 2002, Molecular and Cellular Neuroscience.

[49]  C. Abate-Shen,et al.  BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb. , 2001, Development.

[50]  P. Kingsley,et al.  Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development , 2001, Mechanisms of Development.

[51]  A. Winterpacht,et al.  Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome , 2001, Nature Genetics.

[52]  Antoon F. M. Moorman,et al.  Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[53]  A. Winterpacht,et al.  LMX1B transactivation and expression in nail-patella syndrome. , 2000, Human molecular genetics.

[54]  R J Schwartz,et al.  Evidence for a role of Smad6 in chick cardiac development. , 1999, Developmental biology.

[55]  A. Economides,et al.  Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling. , 1998, Developmental biology.

[56]  W. Scott,et al.  The loss of ventral ectoderm identity correlates with the inability to form an AER in the legless hindlimb bud , 1998, Mechanisms of Development.

[57]  A. Joyner,et al.  Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs. , 1998, Development.

[58]  A. McMahon,et al.  Novel regulatory interactions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutants. , 1997, Development.

[59]  Wolfgang Wurst,et al.  The mouse Engrailed-1 gene and ventral limb patterning , 1996, Nature.

[60]  Juan Carlos Izpisúa Belmonte,et al.  Dorsal cell fate specified by chick Lmxl during vertebrate limb development , 1995, Nature.

[61]  Thomas M. Jessell,et al.  Induction of the LIM homeobox gene Lmx1 by WNT6a establishes dorsoventral pattern in the vertebrate limb , 1995, Cell.

[62]  Andrew P. McMahon,et al.  Dorsalizing signal Wnt-7a required for normal polarity of D–V and A–P axes of mouse limb , 1995, Nature.

[63]  F. Fazal,et al.  PUBLISHED VERSION , 2014 .

[64]  L. Reynard,et al.  The Identification of Transacting Factors That Regulate the Expression of GDF 5 via the Osteoarthritis Susceptibility SNP rs 143383 , 2013 .

[65]  Jean-Michel Claverie,et al.  FusionDB: a database for in-depth analysis of prokaryotic gene fusion events , 2004, Nucleic Acids Res..

[66]  Claude-Alain H. Roten,et al.  Theoretical and practical advances in genome halving , 2004 .

[67]  D. Ovchinnikov,et al.  Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome , 1998, Nature Genetics.

[68]  C. Rodriguez,et al.  Dorsal cell fate specified by chick Lmx1 during vertebrate limb development , 1996, Nature.