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PLOS ONE  2013 

Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs

DOI: 10.1371/journal.pone.0069385

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Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.


[1]  Busser BW, Bulyk ML, Michelson AM (2008) Toward a systems-level understanding of developmental regulatory networks. Curr Opin Genet Dev 18: 521-529. doi:10.1016/j.gde.2008.09.003. PubMed: 18848887.
[2]  Davidson E (2006) The Regulatory Genome: Gene regulatory networks in development and evolution. Academic Press. pp. 304
[3]  Mann RS, Lelli KM, Joshi R (2009) Hox specificity unique roles for cofactors and collaborators. Curr Top Dev Biol 88: 63-101. doi:10.1016/S0070-2153(09)88003-4. PubMed: 19651302.
[4]  Hueber SD, Bezdan D, Henz SR, Blank M, Wu H et al. (2007) Comparative analysis of Hox downstream genes in Drosophila. Development 134: 381-392. doi:10.1242/dev.02746. PubMed: 17166915.
[5]  Busser BW, Shokri L, Jaeger SA, Gisselbrecht SS, Singhania A et al. (2012) Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity. Development 139: 1164-1174. doi:10.1242/dev.077362. PubMed: 22296846.
[6]  Berger MF, Badis G, Gehrke AR, Talukder S, Philippakis AA et al. (2008) Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell 133: 1266-1276. doi:10.1016/j.cell.2008.05.024. PubMed: 18585359.
[7]  Noyes MB, Christensen RG, Wakabayashi A, Stormo GD, Brodsky MH et al. (2008) Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell 133: 1277-1289. doi:10.1016/j.cell.2008.05.023. PubMed: 18585360.
[8]  Bodmer R, Frasch M (2010) Development and aging of the Drosophila heart; N. RosenthalRP Harvey. London: Academic Press.
[9]  Bate M (1993) The mesoderm and its derivatives. In: M. BateA. Martinez Arias. The development of Drosophila melanogaster: Cold Spring Harbor Laboratory Press. pp. 1013-1090.
[10]  Tixier V, Bataillé L, Jagla K (2010) Diversification of muscle types: recent insights from Drosophila. Exp Cell Res 316: 3019-3027. doi:10.1016/j.yexcr.2010.07.013. PubMed: 20673829.
[11]  Baylies MK, Bate M, Ruiz Gomez M (1998) Myogenesis: a view from Drosophila. Cell 93: 921-927. doi:10.1016/S0092-8674(00)81198-8. PubMed: 9635422.
[12]  Jagla K, Bellard M, Frasch M (2001) A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs. Bioessays 23: 125-133. doi:10.1002/1521-1878(200102)23:2. PubMed: 11169585.
[13]  Junion G, Bataillé L, Jagla T, Da Ponte JP, Tapin R et al. (2007) Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors. Genes Dev 21: 3163-3180. doi:10.1101/gad.437307. PubMed: 18056427.
[14]  Jakobsen JS, Braun M, Astorga J, Gustafson EH, Sandmann T et al. (2007) Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network. Genes Dev 21: 2448-2460. doi:10.1101/gad.437607. PubMed: 17908931.
[15]  Liu YH, Jakobsen JS, Valentin G, Amarantos I, Gilmour DT et al. (2009) A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development. Dev Cell 16: 280-291. doi:10.1016/j.devcel.2009.01.006. PubMed: 19217429.
[16]  Capovilla M, Kambris Z, Botas J (2001) Direct regulation of the muscle-identity gene apterous by a Hox protein in the somatic mesoderm. Development 128: 1221-1230. PubMed: 11262224.
[17]  Enriquez J, Boukhatmi H, Dubois L, Philippakis AA, Bulyk ML et al. (2010) Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo. Development 137: 457-466. doi:10.1242/dev.045286. PubMed: 20056681.
[18]  Michelson AM (1994) Muscle pattern diversification in Drosophila is determined by the autonomous function of homeotic genes in the embryonic mesoderm. Development 120: 755-768. PubMed: 7600955.
[19]  Slattery M, Ma L, Négre N, White KP, Mann RS (2011) Genome-wide tissue-specific occupancy of the Hox protein Ultrabithorax and Hox cofactor Homothorax in Drosophila. PLOS ONE 6: e14686. doi:10.1371/journal.pone.0014686. PubMed: 21483663.
[20]  Carrasco-Rando M, Tutor AS, Prieto-Sánchez S, González-Pérez E, Barrios N et al. (2011) Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate. PLOS Genet 7: e1002186. PubMed: 21811416.
[21]  Nose A, Isshiki T, Takeichi M (1998) Regional specification of muscle progenitors in Drosophila: the role of the msh homeobox gene. Development 125: 215-223. PubMed: 9486795.
[22]  Philippakis AA, Busser BW, Gisselbrecht SS, He FS, Estrada B et al. (2006) Expression-guided in silico evaluation of candidate cis regulatory codes for Drosophila muscle founder cells. PLOS Comput Biol 2: e53. doi:10.1371/journal.pcbi.0020053. PubMed: 16733548.
[23]  Berger MF, Philippakis AA, Qureshi AM, He FS, Estep PW 3rd et al. (2006) Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol 24: 1429-1435. doi:10.1038/nbt1246. PubMed: 16998473.
[24]  Gallo SM, Gerrard DT, Miner D, Simich M, Des Soye B et al. (2011) REDfly v3.0: toward a comprehensive database of transcriptional regulatory elements in Drosophila. Nucleic Acids Res 39: D118-D123. doi:10.1093/nar/gkq999. PubMed: 20965965.
[25]  Robasky K, Bulyk ML (2011) UniPROBE, update 2011: expanded content and search tools in the online database of protein-binding microarray data on protein-DNA interactions. Nucleic Acids Res 39: D124-D128. doi:10.1093/nar/gkq992. PubMed: 21037262.
[26]  Busser BW, Taher L, Kim Y, Tansey T, Bloom MJ et al. (2012) A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis. PLOS Genet 8: e1002531.
[27]  Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40: 476-483. doi:10.1038/ng.101. PubMed: 18311141.
[28]  Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166: 1775-1782. doi:10.1534/genetics.166.4.1775. PubMed: 15126397.
[29]  Galant R, Walsh CM, Carroll SB (2002) Hox repression of a target gene: extradenticle-independent, additive action through multiple monomer binding sites. Development 129: 3115-3126. PubMed: 12070087.
[30]  Gebelein B, Culi J, Ryoo HD, Zhang W, Mann RS (2002) Specificity of Distalless repression and limb primordia development by abdominal Hox proteins. Dev Cell 3: 487-498. doi:10.1016/S1534-5807(02)00257-5. PubMed: 12408801.
[31]  Grienenberger A, Merabet S, Manak J, Iltis I, Fabre A et al. (2003) Tgfbeta signaling acts on a Hox response element to confer specificity and diversity to Hox protein function. Development 130: 5445-5455. doi:10.1242/dev.00760. PubMed: 14507783.
[32]  Hersh BM, Carroll SB (2005) Direct regulation of knot gene expression by Ultrabithorax and the evolution of cis-regulatory elements in Drosophila. Development 132: 1567-1577. doi:10.1242/dev.01737. PubMed: 15753212.
[33]  Hersh BM, Nelson CE, Stoll SJ, Norton JE, Albert TJ et al. (2007) The UBX-regulated network in the haltere imaginal disc of D. melanogaster. Dev Biol 302: 717-727. doi:10.1016/j.ydbio.2006.11.011. PubMed: 17174297.
[34]  Ryoo HD, Mann RS (1999) The control of trunk Hox specificity and activity by Extradenticle. Genes Dev 13: 1704-1716. doi:10.1101/gad.13.13.1704. PubMed: 10398683.
[35]  Zhu X, Ahmad SM, Aboukhalil A, Busser BW, Kim Y et al. (2012) Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors. Development 139: 1457-1466. doi:10.1242/dev.069005. PubMed: 22378636.
[36]  Jin H, Stojnic R, Adryan B, Ozdemir A, Stathopoulos A et al. (2013) Genome-wide screens for in vivo tinman binding sites identify cardiac enhancers with diverse functional architectures. PLOS Genet 9: e1003195. PubMed: 23326246.
[37]  Bourgouin C, Lundgren SE, Thomas JB (1992) apterous is a Drosophila LIM domain gene required for the development of a subset of embryonic muscles. Neuron 9: 549-561. doi:10.1016/0896-6273(92)90192-G. PubMed: 1524829.
[38]  Jagla T, Bidet Y, Da Ponte JP, Dastugue B, Jagla K (2002) Cross-repressive interactions of identity genes are essential for proper specification of caridiac and muscular fates in Drosophila. Development 129: 1037-1047.
[39]  Müller D, Jagla T, Bodart LM, J?hrling N, Dodt HU et al. (2010) Regulation and functions of the lms homeobox gene during development of embryonic lateral transverse muscles and direct flight muscles in Drosophila. PLOS ONE 5: e14323. doi:10.1371/journal.pone.0014323. PubMed: 21179520.
[40]  Clark IB, Boyd J, Hamilton G, Finnegan DJ, Jarman AP (2006) D-six4 plays a key role in patterning cell identities deriving from the Drosophila mesoderm. Dev Biol 294: 220-231. doi:10.1016/j.ydbio.2006.02.044. PubMed: 16595131.
[41]  Azpiazu N, Frasch M (1993) tinman and bagpipe -- two homeobox genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev 7: 1325-1340. doi:10.1101/gad.7.7b.1325. PubMed: 8101173.
[42]  Bodmer R (1993) The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118: 719-729. PubMed: 7915669.
[43]  Zinzen RP, Girardot C, Gagneur J, Braun M, Furlong EE (2009) Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature 462: 65-70. doi:10.1038/nature08531. PubMed: 19890324.
[44]  Siggers T, Duyzend MH, Reddy J, Khan S, Bulyk ML (2011) Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex. Mol Syst Biol 7: 555. PubMed: 22146299.
[45]  Slattery M, Riley T, Liu P, Abe N, Gomez-Alcala P et al. (2011) Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell 147: 1270-1282. doi:10.1016/j.cell.2011.10.053. PubMed: 22153072.
[46]  Schaub C, Nagaso H, Jin H, Frasch M (2012) Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification. Development 139: 1001-1012. doi:10.1242/dev.073890. PubMed: 22318630.
[47]  Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y et al. (2001) Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat Genet 28: 276-280. doi:10.1038/90123. PubMed: 11431700.
[48]  Stennard FA, Costa MW, Elliott DA, Rankin S, Haast SJ et al. (2003) Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev Biol 262: 206-224. doi:10.1016/S0012-1606(03)00385-3. PubMed: 14550786.
[49]  Junion G, Spivakov M, Girardot C, Braun M, Gustafson EH et al. (2012) A transcription factor collective defines cardiac cell fate and reflects lineage history. Cell 148: 473-486. doi:10.1016/j.cell.2012.01.030. PubMed: 22304916.
[50]  Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J et al. (2003) The formation of skeletal muscle: from somite to limb. J Anat 202: 59-68. doi:10.1046/j.1469-7580.2003.00139.x. PubMed: 12587921.
[51]  Niro C, Demignon J, Vincent S, Liu Y, Giordani J et al. (2010) Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome. Dev Biol 338: 168-182. doi:10.1016/j.ydbio.2009.11.031. PubMed: 19962975.
[52]  Richard AF, Demignon J, Sakakibara I, Pujol J, Favier M et al. (2011) Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression. Dev Biol 359: 303-320. doi:10.1016/j.ydbio.2011.08.010. PubMed: 21884692.
[53]  Moens CB, Selleri L (2006) Hox cofactors in vertebrate development. Dev Biol 291: 193-206. doi:10.1016/j.ydbio.2005.10.032. PubMed: 16515781.
[54]  Chang CP, Brocchieri L, Shen WF, Largman C, Cleary ML (1996) Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus. Mol Cell Biol 16: 1734-1745. PubMed: 8657149.
[55]  In der Rieden PM, Mainguy G, Woltering JM, Durston AJ (2004) Homeodomain to hexapeptide or PBC-interaction-domain distance: size apparently matters. Trends Genet 20: 76-79. doi:10.1016/j.tig.2003.12.001. PubMed: 14746988.
[56]  Grove CA, De Masi F, Barrasa MI, Newburger DE, Alkema MJ et al. (2009) A multiparameter network reveals extensive divergence between C. elegans bHLH transcription factors. Cell 138: 314-327. doi:10.1016/j.cell.2009.04.058. PubMed: 19632181.
[57]  Landschulz WH, Johnson PF, McKnight SL (1988) The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240: 1759-1764. doi:10.1126/science.3289117. PubMed: 3289117.
[58]  Mahaffey JW (2005) Assisting Hox proteins in controlling body form: are there new lessons from flies (and mammals)? Curr Opin Genet Dev 15: 422-429. doi:10.1016/j.gde.2005.06.009. PubMed: 15979870.
[59]  Dasen JS, De Camilli A, Wang B, Tucker PW, Jessell TM (2008) Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell 134: 304-316. doi:10.1016/j.cell.2008.06.019. PubMed: 18662545.
[60]  Gebelein B, McKay DJ, Mann RS (2004) Direct integration of Hox and segmentation gene inputs during Drosophila development. Nature 431: 653-659. doi:10.1038/nature02946. PubMed: 15470419.
[61]  Zaret KS, Carroll JS (2011) Pioneer transcription factors: establishing competence for gene expression. Genes Dev 25: 2227-2241. doi:10.1101/gad.176826.111. PubMed: 22056668.
[62]  Busser BW, Huang D, Rogacki KR, Lane EA, Shokri L et al. (2012) Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network. Proc Natl Acad Sci U S A 109: 20768-20773. doi:10.1073/pnas.1210415109. PubMed: 23184988.


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