Mle (DHX9) helicase regulates the expression of constitutive and inducible isoforms of the conserved nuclear receptor FTZ-F1 (NR5A3)

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

In addition to participating in dosage compensation, the MLE helicase in D. melanogaster performs many functions in the regulation of gene expression, as does its human ortholog DHX9. Many of these functions are evolutionarily conserved and poorly explored. MLE has previously been shown to be involved in the regulation of inducible transcription of the ftz-f1 gene encoding the evolutionarily conserved nuclear receptor NR5A3. The ftz-f1 gene also encodes a constitutive transcript synthesized from an alternative promoter. The present work is devoted to the investigation of the role of MLE in the regulation of constitutive transcription of the ftz-f1 gene. This work shows that in S2 cell culture, MLE binds to the constitutive promoter and controls both inducible and constitutive transcription of the ftz-f1 gene. A novel MLE-binding cis-regulatory element of the ftz-f1 gene, enhancer 663, was identified. Using chromosome conformation capture technique the interaction of enhancer 663 with constitutive and inducible promoters of ftz-f1 gene in S2 cell culture was demonstrated. Examination of enhancer 663 histone H3 acetylation showed that it is involved in the activity of both promoters. Knockdown of MLE in S2 cell culture causes an increase in constitutive transcription. The effect of MLE on transcription beyond dosage compensation in vivo at the adult stage was shown for the first time. It was shown that at the adult stage MLE binds to both inducible and constitutive promoters and to enhancer 663. Mutation in the mle gene leads to increased expression of both transcripts of the ftz-f1 gene in females. The data obtained are important for understanding and further study of the evolutionarily conserved functions of MLE and its human ortholog DHX9.

Full Text

Restricted Access

About the authors

J. V. Nikolenko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: julia.v.nikolenko@gmail.com
Russian Federation, Moscow

S. G. Georgieva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: julia.v.nikolenko@gmail.com
Russian Federation, Moscow

References

  1. Lee C.G., Hurwitz J. (1993) Human RNA helicase A is homologous to the maleless protein of Drosophila. J. Biol. Chem. 268, 16822–16830.
  2. Lee T., Pelletier J. (2016) The biology of DHX9 and its potential as a therapeutic target. Oncotarget. 7, 42716–42739.
  3. Calame D.G., Guo T., Wang C., Garrett L., Jolly A., Dawood M., Kurolap A., Henig N.Z., Fatih J.M., Herman I., Du H., Mitani T., Becker L., Rathkolb B., Gerlini R., Seisenberger C., Marschall S., Hunter J.V., Gerard A., Heidlebaugh A., Challman T., Spillmann R.C., Jhangiani S.N., Coban-Akdemir Z., Lalani S., Liu L., Revah-Politi A., Iglesias A., Guzman E., Baugh E., Boddaert N., Rondeau S., Ormieres C., Barcia G., Tan Q.K.G., Thiffault I., Pastinen T., Sheikh K., Biliciler S., Mei D., Melani F., Shashi V., Yaron Y., Steele M., Wakeling E., Østergaard E., Nazaryan-Petersen L.; Undiagnosed Diseases Network; Millan F., Santiago-Sim T., Thevenon J., Bruel A.L., Thauvin-Robinet C., Popp D., Platzer K., Gawlinski P., Wiszniewski W., Marafi D., Pehlivan D., Posey J.E., Gibbs R.A., Gailus-Durner V., Guerrini R., Fuchs H., Hrabě de Angelis M., Hölter S.M., Cheung H.H., Gu S., Lupski J.R. (2023) Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease. Am. J. Hum. Genet. 110, 1394–1413.
  4. Gulliver C., Hoffmann R., Baillie G.S. (2020) The enigmatic helicase DHX9 and its association with the hallmarks of cancer. Future Sci. OA. 7, FSO650.
  5. Kotlikova I.V., Demakova O.V., Semeshin V.F., Shloma V.V., Boldyreva L.V., Kuroda M.I., Zhimulev I.F. (2006) The Drosophila dosage compensation complex binds to polytene chromosomes independently of developmental changes in transcription. Genetics. 172, 963–974.
  6. Cugusi S., Li Y., Jin P., Lucchesi J.C. (2016) The Drosophila helicase MLE targets hairpin structures in genomic transcripts. PLoS Genet. 12, e1005761.
  7. Cugusi S., Kallappagoudar S., Ling H., Lucchesi J.C. (2015) The Drosophila helicase maleless (MLE) is implicated in functions distinct from its role in dosage compensation. Mol. Cell. Proteomics. 14, 1478–1488.
  8. Николенко Ю.В., Куршакова М.М., Краснов А.Н. (2019) Мультифункциональный белок ENY2 взаимодействует с РНК-хеликазой MLE. Докл. Акад. Наук. 489, 637–640.
  9. Николенко Ю.В., Куршакова М.М., Краснов%А.Н., Георгиева С.Г. (2021) Хеликаза MLE – новый участник регуляции транскрипции гена ftz-f1, кодирующего ядерный рецептор у высших эукариот. Докл. Акад. Наук. Науки о жизни. 496, 48–51.
  10. Beachum A.N., Whitehead K.M., McDonald S.I., Phipps D.N., Berghout H.E., Ables E.T. (2021) Orphan nuclear receptor ftz-f1 (NR5A3) promotes egg chamber survival in the Drosophila ovary. G3 (Bethesda). 11, jkab003.
  11. Hughes C.H.K., Smith O.E., Meinsohn M.C., Brunelle M., Gévry N., Murphy B.D. (2023) Steroidogenic factor 1 (SF-1; Nr5a1) regulates the formation of the ovarian reserve. Proc. Natl. Acad. Sci. USA. 120, e2220849120.
  12. Ueda H., Sonoda S., Lesley Brown J., Scott M.P., Wu C. (1990) A sequence-specific DNA-binding protein that activates fushi tarazu segmentation gene expression. Genes Dev. 4, 624–635.
  13. Vorobyeva N.E., Nikolenko J.V., Nabirochkina E.N., Krasnov A.N., Shidlovskii Y.V., Georgieva S.G. (2012) SAYP and Brahma are important for ‘repressive’ and ‘transient’ Pol II pausing. Nucl. Acids Res. 40, 7319–7331.
  14. Lavorgna G., Karim F.D., Thummel C.S., Wu C. (1993) Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis. Proc. Natl. Acad. Sci. USA. 90, 3004–3008.
  15. Ohno C.K., Ueda H., Petkovich M. (1994) The Drosophila nuclear receptors FTZ-Flα and FTZ-F1β compete as monomers for binding to a site in the fushi tarazu gene. Mol. Cell Biol. 14, 3166–3175.
  16. Yu Y., Li W., Su K., Yussa M., Han W., Perrimon N., Pick L. (1997) The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz. Nature. 385, 552–555.
  17. Broadus J., McCabe J.R., Endrizzi B., Thummel C.S., Woodard C.T. (1999) The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone. Mol. Cell. 3, 143–149.
  18. Woodard C.T., Baehrecke E.H., Thummel C.S. (1994) A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell. 79, 607–615.
  19. Boulanger A., Clouet-Redt C., Farge M., Flandre A., Guignard T., Fernando C., Juge F., Dura J.M. (2011) ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling. Nat. Neurosci. 14, 37–46.
  20. Yamada M.A., Murata T., Hirose S., Lavorgna G., Suzuki E., Ueda H. (2000) Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster. Development. 127, 5083–5092.
  21. Clemens J.C., Worby C.A., Simonson-Leff N., Muda M., Maehama T., Hemmings B.A., Dixon J.E. (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA. 97, 6499–6503.
  22. Гаврилов А.А., Разин С.В. (2008) Изучение пространственной организации домена альфа-глобиновых генов кур методом 3С. Биохимия. 73, 1486–1494.
  23. Arnold C.D., Gerlach D., Stelzer C., Boryń Ł.M., Rath M., Stark A. (2013) Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science. 339, 1074–1077.
  24. Yáñez-Cuna J.O., Arnold C.D., Stampfel G., Borýn Ł.M., Gerlach D., Rath M., Stark A. (2014) Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features. Genome Res. 24, 1147–1156.
  25. de Almeida B.P., Reiter F., Pagani M., Stark A. (2022) DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of synthetic enhancers. Nat. Genet. 54, 613–624.
  26. Heintzman N.D., Stuart R.K., Hon G., Fu Y., Ching C.W., Hawkins R.D., Barrera L.O., Van Calcar S., Qu C., Ching K.A., Wang W., Weng Z., Green R.D., Crawford G.E., Ren B. (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318.
  27. Koenecke N., Johnston J., Gaertner B., Natarajan M., Zeitlinger J. (2016) Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis. Genome Biol. 17, 196.
  28. Ong C.T., Corces V.G. (2011) Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat. Rev. Genet. 12, 283–293.
  29. Cubenãs-Potts C., Rowley M.J., Lyu X., Li G., Lei E.P., Corces V.G. (2017) Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucl. Acids Res. 45, 1714–1730.
  30. Marsman J., Horsfield J.A. (2012) Long distance relationships: enhancer-promoter communication and dynamic gene transcription. Biochim. Biophys. Acta. 1819, 1217–1227.
  31. Ашниев Г.А., Георгиева С.Г., Николенко Ю.В. (2024) Функции хеликазы MLE Drosophila melanogaster вне дозовой компенсации: молекулярная природа и плейотропный эффект мутации mle[9]. Генетика. 60, 34–46.
  32. Николенко Ю.В., Георгиева С.Г., Копытова Д.В. (2023) Разнообразие функций хеликазы MLE в регуляции экспрессии генов у высших эукариот. Молекуляр. биология. 57, 10–23.
  33. Николенко Ю.В., Краснов А.Н., Воробьева Н.Е. (2019) Ремоделирующий хроматин комплекс SWI/SNF влияет на пространственную организацию локуса гена ftz-f1. Генетика. 55, 156–164.
  34. Николенко Ю.В., Краснов А.Н., Мазина М.Ю., Георгиева С.Г., Воробьева Н.Е. (2017) Изучение свойств нового экдизонзависимого энхансера. Докл. Акад. Наук. 474, 756–759.
  35. Mazina M.Yu., Kovalenko E.V., Derevyanko P.K., Nikolenko J.V., Krasnov A.N., Vorobyeva N.E. (2018) One signal stimulates different transcriptional activation mechanisms. Biochim. Biophys. Acta (BBA) – Gene Regulatory Mechanisms. 1861, 178–189.
  36. Mazina M.Y., Nikolenko J.V, Fursova N.A., Nedil’ko P.N., Krasnov A.N., Vorobyeva N.E. (2015) Early-late genes of the ecdysone cascade as models for transcriptional studies. Cell Cycle. 14, 3593–3601.
  37. Vorobyeva N.E., Nikolenko J.V., Krasnov A.N., Kuzmina J.L., Panov V.V., Nabirochkina E.N., Georgieva S.G., Shidlovskii Y.V. (2011) SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation. Cell Cycle. 10, 1821–1827.
  38. Samata M., Akhtar A. (2018) Dosage compensation of the X chromosome: a complex epigenetic assignment involving chromatin regulators and long noncoding RNAs. Annu. Rev. Biochem. 87, 323–350.
  39. McDowell K.A., Hilfiker A, Lucchesi J.C. (1996) Dosage compensation in Drosophila: the X chromosome binding of MSL-1 and MSL-2 in female embryos is prevented by the early expression of the Sxl gene. Mech. Dev. 57,113–119.
  40. Aratani S., Kageyama Y., Nakamura A., Fujita H., Fujii R., Nishioka K., Nakajima T. (2008) MLE activates transcription via the minimal transactivation domain in Drosophila. Int. J. Mol. Med. 21, 469–476.
  41. Knapp E.M., Li W., Singh V., Sun J. (2020) Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim. Elife. 9, e54568.
  42. Meinsohn M.C., Smith O.E., Bertolin K., Murphy B.D. (2019) The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiol. Rev. 99, 1249–1279.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. The structure of the ftz-f1 gene and its expression in the S2 cells of D. melanogaster. a – Schematic representation of the ftz-f1 gene and its transcripts: constitutive ftz-f1-B and inducible ftz-f1-C. Exons are indicated by gray rectangles, the coding region is highlighted in a lighter color, arrows indicate the transcription start sites, the asterisk shows the position of the enhancer in the third intron. Black segments (1–7) indicate the position of the primers, B and C – “anchor” primers used for 3C analysis of the spatial structure of the gene. b – Changes in the expression level of the constitutive and inducible transcripts of the ftz-f1 gene in the model system in S2 cells of D. melanogaster. Here and below: “–” – in the medium without the addition of ecdysone, “+” – after incubation in the medium with 0.3 μM ecdysone for 16 h, “+;-” – after washing and subsequent incubation in the medium without ecdysone for 3 h. Here and below: the expression level of the ftz-f1 gene was normalized to the expression level of the control ras64B gene. The expression level is presented in relative units, the ftz-f-C level at the “–” stage is taken as 1. c – Changes in the expression level of the constitutive ftz-f-B transcript in the model system in S2 cells against the background of MLE knockdown by RNA interference. The expression level is presented in relative units, the ftz-f-B level in the control at the “–” stage is taken as 1.

Download (168KB)
Indexing metadata
3. Fig. 2. Study of enhancer 663 in a model system in D. melanogaster S2 cells by chromatin immunoprecipitation. a – Analysis of MLE binding to enhancer 663 and promoters of constitutive and inducible transcripts of the ftz-f1 gene. “+1600” is a control point with a low level of MLE binding. b – Content (%) of histone H3 modifications relative to the total amount of histone H3 on enhancer 663. H3me1K4 – histone H3, monomethylated at lysine residue 4; H3AcK27 – histone H3, acetylated at lysine residue 27.

Download (86KB)
Indexing metadata
4. Fig. 3. Analysis of the interaction of enhancer 663 with the constitutive (a) and inducible (b) promoters of the ftz-f1 gene by the chromosome conformation fixation method (3C). Exons are indicated by gray rectangles, the coding region is highlighted in lighter color; arrows indicate the transcription start sites; an asterisk shows the position of enhancer 663; segments (1–7) indicate the position of the primers, black columns B and C – the position of the “anchor” primers. The distance is given in bp relative to the transcription start site. The ligation frequency is given in relative units, the ligation frequency of enhancer 663 with promoter B (a) and promoter C (b), respectively, at the “–” stage is taken as 1.

Download (134KB)
Indexing metadata
5. Fig. 4. Study of the effect of MLE on the expression of the ftz-f1 gene in vivo in D. melanogaster females at the imago stage. a – Changes in the expression level of the constitutive and inducible transcripts of the ftz-f1 gene against the background of the mle[9] mutation. wt – wild type. The expression level is presented in relative units, the expression level of the control gene ras64B is taken as 1. b – Analysis of binding of the mutant version of the MLE protein (mle[9]) and the wild-type protein (wt) to the 663 enhancer, constitutive and inducible promoters of the ftz-f1 gene by chromatin immunoprecipitation. “+1600” is a control point with a low level of MLE binding.

Download (142KB)
Indexing metadata

Copyright (c) 2025 Russian Academy of Sciences