Analysis of whole-genome binding patterns of GAGA and CNC transcription factors during Drosophila melanogaster development

On the basis of available data of ChIP-seq and ChIPchip experiments performed using antibodies against GAGA and CNC transcription factors, genome-wide binding mapping of these factors at hours 0–12 and 16–24 of Drosophila embryogenesis, as well as on white pre-pupae stage, was conducted. It was shown that the bulk of GAGA and CNC binding falls into promoter regions and introns, with the maximal density of peaks in the vicinity of the transcription start site. Moreover, in both 0–12 and 16–24 hour old embryos GAGA and CNC are frequently co-localized, while on white pre-pupae stage there is no co-localization of these factors on a genome–wide scale. In order to select a set of genes potentially co-regulated by GAGA and CNC, the study of their co-binding in annotated regulatory regions (promoter areas and segments corresponding to the 5’-UTR and 3’-UTR of mRNA) was performed. The results obtained clearly demonstrated that the sets of genes characterized by co-binding of both factors vary greatly at different stages. Thus from 353 genes with overlapped GAGA and CNC binding loci on the 0–12 hour old embryos and 611 genes on the 0–12 hour old embryos only 61 genes “belong” to both stages. For an explanation it is proposed that different sets of target genes are regulated by combinations of various GAGA and CNC isoforms, which are characterized by distinct expression patterns during drosophila embryogenesis. Functional annotation analysis of genes, in whose regulatory regions both GAGA and CNC were found at all investigated stages, demonstrates enrichment by genes controlling embryogenesis, neurogenesis and wing development. The data obtained suggest the interaction of GAGA and CNC during D. melanogaster embryogenesis.

1. Adryan B., Teichmann S.A. The developmental expression dynamics of Drosophila melanogaster transcription factors. Genome Biol. 2010; 11(4):R40.

2. Arnold C.D., Gerlach D., Stelzer C., Boryn L.M., Rath M., Stark A. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science. 2013;339(6123):1074-1077.

3. Benyajati C., Mueller L., Xu N., Pappano M., Gao J., Mosammaparast M., Conklin D., Granok H., Craig, C., Elgin S. Multiple isoforms of GAGA factor, a critical component of chromatin structure. Nucl. Acids Res. 1997;25(16):3345-3353.

4. Bhat K.M., Farkas G., Karch F., Gyurkovics H., Gausz J., Schedl P. The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division. Development (Cambridge, England). 1996;122(4):1113-1124.

5. Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics (Oxford, England). 2014; 30(15):2114-2120.

6. Bulger M., Groudine M. Functional and mechanistic diversity of distal transcription enhancers. Cell. 2011;144(3):327-339.

7. Campos-Ortega J.A., Hartenstein V. The Embryonic Development of Drosophila Melanogaster. Berlin; Heidelberg: Springer, 1997.

8. Charoensawan V., Wilson D., Teichmann S.A. Genomic repertoires of DNA-binding transcription factors across the tree of life. Nucl. Acids Res. 2010;38(21):7364-7377.

9. Diehl A.G., Boyle A.P. Deciphering ENCODE. Trends Genet. 2016; 32(4):238-249.

10. Espinas M.L., Jimenez-Garcia E., Vaquero A., Canudas S., Bernues J., Azorin F. The N-terminal POZ domain of GAGA mediates the formation of oligomers that bind DNA with high affinity and specificity. J. Biol. Chemistry. 1999;274:16461-16469.

11. Farkas G., Gausz J., Galloni M., Reuter G., Gyurkovics H., Karch F. The Trithorax-like gene encodes the Drosophila GAGA factor. Nature. 1994;371(6500):806-808.

12. Farnham P.J. Insights from genomic profiling of transcription factors. Nat. Rev. Genet. 2009;10(9):605-616.

13. Filion G.J., van Bemmel J.G., Braunschweig U., Talhout W., Kind J., Ward L.D., Brugman W., de Castro I.J., Kerkhoven R.M., Bussemaker H.J., van Steensel B. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell. 2010; 143(2):212- 224.

14. Frith M.C., Valen E., Krogh A., Hayashizaki Y., Carninci P., Sandelin A. A code for transcription initiation in mammalian genomes. Genome Res. 2008;18(1):1-12.

15. Granok H., Leibovitch B.A., Shaffer C.D., Elgin S.C. Chromatin. Gaga over GAGA factor. Curr. Biol. 1995;5(3):238-241.

16. Hammonds A.S., Bristow C.A., Fisher W.W., Weiszmann R., Wu S., Hartenstein V., Kellis M., Yu B., Frise E., Celniker S.E. Spatial expression of transcription factors in Drosophila embryonic organ development. Genome Biol. 2013;14(12):R140.

17. Harbison C.T., Gordon D.B., Lee T.I., Rinaldi N.J., Macisaac K.D., Danford T.W., Hannett N.M., Tagne J.-B., Reynolds D.B., Yoo J., Jennings E.G., Zeitlinger J., Pokholok D.K., Kellis M., Rolfe P.A., Takusagawa K.T., Lander E.S., Gifford D.K., Fraenkel E., Young R.A. Transcriptional regulatory code of a eukaryotic genome. Nature. 2004;431(7004):99-104.

18. Hoskins R.A., Carlson J.W., Wan K.H., Park S., Mendez I., Galle S.E., Booth B.W., Pfeiffer B.D., George R.A., Svirskas R., Krzywinski M., Schein J., Accardo M.C., Damia E., Messina G., Méndez-Lago M., de Pablos B., Demakova O.V., Andreyeva E.N., Boldyreva L.V., Marra M., Carvalho A.B., Dimitri P., Villasante A., Zhimulev I.F., Rubin G.M., Karpen G.H., Celniker S.E. The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 2015;25(3):445-458.

19. Ingham P.W. The molecular genetics of embryonic pattern formation in Drosophila. Nature. 1988;335(6185):25-34.

20. Iyer E.P.R., Iyer S.C., Sullivan L., Wang D., Meduri R., Graybeal L.L., Cox D.N. Functional genomic analyses of two morphologically distinct classes of Drosophila sensory neurons: post-mitotic roles of transcription factors in dendritic patterning. PloS ONE. 2013;8(8):e72434.

21. Karagodin D.A., Omelina E.S., Fedorova E.V., Baricheva E.M. Identification of functionally significant elements in the second intron of the Drosophila melanogaster Trithorax-like gene. Gene. 2013;520(2): 178-184.

22. Kvon E.Z., Stampfel G., Yanez-Cuna J.O., Dickson B.J., Stark A. HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature. Genes Develop. 2012;26(9): 908-913.

23. Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9(4):357-359.

24. Lelli K.M., Slattery M., Mann R.S. Disentangling the many layers of eukaryotic transcriptional regulation. Ann. Rev. Genet. 2012;46(1):43-68.

25. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. & 1000 Genome Project Data Processing Subgroup The Sequence Alignment/Map format and SAMtools. Bioinformatics (Oxford, England). 2009;25(16):2078-2079.

26. McGinnis N., Ragnhildstveit E., Veraksa A., McGinnis W. A cap “n” collar protein isoform contains a selective Hox repressor function. Development (Cambridge, England). 1998;125(22):4553-4564.

27. Merkulova T.I., Ananko E.A., Ignat’eva E.V., Kolchanov N.A. Regulatory transcription codes in eukaryotic genomes. Genetika = Genetics (Moscow). 2013;49(1):37-54. (in Russian)

28. Mohler J., Mahaffey J.W., Deutsch E., Vani K. Control of Drosophila head segment identity by the bZIP homeotic gene cnc. Development (Cambridge, England). 1995;121(1):237-247.

29. Moorman C., Sun L.V., Wang J., de Wit E., Talhout W., Ward L.D., Greil F., Lu X.-J., White K.P., Bussemaker H.J., van Steensel B. Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster. Proc. Natl. Acad. Sci. 2006;103:12027-12032.

30. Nègre N., Brown C.D., Ma L., Bristow C.A., Miller S.W., Wagner U., … White K.P. A cis- regulatory map of the Drosophila genome. Nature. 2011;471(7339):527-531. http://doi.org/10.1038/nature09990.

31. Nègre N., Brown C.D., Shah P.K., Kheradpour P., Morrison C.A., Henikoff J.G., … White K.P. A comprehensive map of insulator elements for the Drosophila genome. PLoS Genetics. 2010;6(1): e1000814. http://doi.org/10.1371/journal.pgen.1000814.

32. O’Brien T., Wilkins R.C., Giardina C., Lis J.T. Distribution of GAGA protein on Drosophila genes in vivo. Genes Development. 1995;9(9): 1098-1110.

33. Ochoa-Espinosa A., Small S. Developmental mechanisms and cis-regulatory codes.Curr. Opin. Gen. Dev. 2006;16(2):165-170.

34. Pagans S., Ortiz-Lombardia M., Espinas M.L., Bernues J., Azorin F. The Drosophila transcription factor tramtrack (TTK) interacts with Trithorax-like (GAGA) and represses GAGA-mediated activation. Nucl. Acids Res. 2002;30(20):4406-4413.

35. Perelygina L.M., Baricheva E.M., Sebeleva T.E., Kokoza V.A. The evolutionarily conserved gene Nc70F expressed in nerve tissue of Drosophila melanogaster encodes a protein homologous to the mouse delta transcription factor. Genetika = Genetics (Moscow). 1993;29(10):1597-1607.

36. Rivera-Pomar R., Jãckle H. From gradients to stripes in Drosophila embryogenesis: filling in the gaps. Trends Genet. 1996;12(11):478-483.

37. Roy S., Ernst J., Kharchenko P.V., Kheradpour P., Negre N., Eaton M.L., Landolin J.M., Bristow C.A., Ma L.J., Lin M.F., … Kellis M. Iden-tification of functional elements and regulatory circuits by Drosophila modENCODE. Science. 2010;330(6012):1787-1797.

38. Schertel C., Albarca M., Rockel-Bauer C., Kelley N.W., Bischof J., Hens K., van Nimwegen E., Basler K., Deplancke B. A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development. Genome Res. 2015;25(4):514-523.

39. Segal E., Raveh-Sadka T., Schroeder M., Unnerstall U., Gaul U. Predicting expression patterns from regulatory sequence in Drosophila segmentation. Nature. 2008;451(7178):535-540.

40. Slattery M., Ma L., Spokony R.F., Arthur R.K., Kheradpour P., Kundaje A., Nègre N., Crofts A., Ptashkin R., Zieba J., Ostapenko A., Suchy S., Victorsen A., Jameel N., Grundstad A.J., Gao W., Moran J.R., Rehm E.J., Grossman R.L., Kellis M., White K.P. Diverse patterns of genomic targeting by transcriptional regulators in Drosophila melanogaster. Genome Res. 2014;24(7):1224-1235.

41. Soeller W.C., Oh C.E., Kornberg T.B. Isolation of cDNAs encoding the Drosophila GAGA transcription factor. Mol. Cell. Biol. 1993; 13(12):7961-7970.

42. Tomancak P., Ohler U. Mapping the complexity of transcription control in higher eukaryotes. Genome Biol. 2010;11(4):115.

43. Veraksa A., McGinnis N., Li X., Mohler J., McGinnis W. Cap “n” collar B cooperates with a small Maf subunit to specify pharyngeal development and suppress deformed homeotic function in the Drosophila head. Development (Cambridge, England). 2000;127(18): 4023- 4037.

44. Zhang Y., Liu T., Meyer C.A., Eeckhoute J., Johnson D.S., Bernstein B.E., Nusbaum C., Myers R.M., Brown M., Li W., Liu X.S. Modelbased analysis of ChIP-Seq (MACS). Genome Biol. 2008;9(9):R137.