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ELAV mediates the repression of abd-A by transcriptional interference in the embryonic CNS of Drosophila

CASTRO ALVAREZ, Javier José

Abstract

The iab-8 ncRNA of Drosophila melanogaster had previously been described as a repressor of the Hox gene abd-A in parasegment 13 of the embryonic central nervous system via both the production of a microRNA and a second, cis- acting mechanism, which was proposed to be transcriptional interference. Using genetic reporters to reproduce the interaction between the iab-8 ncRNA and abd-A, we confirmed this hypothesis. This mechanism is most likely dependent on the production of a CNS-specific isoform of the iab-8 ncRNA that invades the transcriptional unit of abd-A. The generation of this isoform relies on the activity of the RNA-binding protein ELAV that mediates alternative splicing of genes produced in neurons.

Failure in the production of these transcripts in elav mutants causes derepression of abd-A in PS13 of the CNS. Based on these findings, we propose that ELAV mediates the repression of abd-A by transcriptional interference in the embryonic CNS of Drosophila.

CASTRO ALVAREZ, Javier José. ELAV mediates the repression of abd-A by

transcriptional interference in the embryonic CNS of Drosophila. Thèse de doctorat : Univ. Genève, 2019, no. Sc. Vie 29

DOI : 10.13097/archive-ouverte/unige:124130 URN : urn:nbn:ch:unige-1241309

Available at:

http://archive-ouverte.unige.ch/unige:124130

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de Biologie

Département de Génétique et Evolution Professeur François Karch

ELAV Mediates the Repression of abd-A by Transcriptional Interference in the Embryonic CNS of Drosophila

THÈSE

présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,

mention Biosciences moléculaires

par

Javier José CASTRO ALVAREZ de

Venezuela

Thèse No 29

GENÈVE

Atelier d’Impression Numérique - Repromail 2019

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TABLE OF CONTENTS

TABLE  OF  CONTENTS  ...  1  

ACKNOWLEDGEMENTS  ...  4  

FIGURE  INDEX  ...  8  

RÉSUMÉ  ...  10  

ABSTRACT  ...  14  

INTRODUCTION  ...  18  

Hox  genes  ...  18  

The  Bithorax  Complex  of  Drosophila  melanogaster  ...  20  

Ed  Lewis  and  the  BX-­‐C  ...  20  

The  Bithorax  Complex  Hox  genes  ...  21  

Regulation  in  the  BX-­‐C:  The  "open  for  business"  model  ...  23  

Cis-­‐regulatory  domains  of  the  BX-­‐C  ...  26  

Initiator  sequences  ...  27  

Boundaries  ...  28  

Maintenance  elements  ...  28  

Intergenic  RNAs  in  the  Bithorax  Complex  ...  29  

The  early  domain-­‐specific  transcription  ...  29  

The  noncoding  RNA  bithoraxoid  (bxd)  ...  30  

The  iab-­‐4  noncoding  RNA  ...  30  

The  iab-­‐8  noncoding  RNA  ...  30  

The  msa  transcript  ...  32  

Regulation  of  abdominal-­‐A  by  the  iab-­‐8  ncRNA  ...  34  

The  mir-­‐iab-­‐8  microRNA  ...  36  

The  production  of  mir-­‐iab-­‐8  is  not  the  only  function  of  the  iab-­‐8  ncRNA  ...  36  

Evolutionary  conservation  of  the  iab-­‐8  ncRNA  ...  40  

Transcriptional  interference  as  a  regulatory  mechanism  ...  41  

Transcriptional  interference  in  Hox  clusters  ...  44  

OBJECTIVES  ...  46  

MATERIALS  AND  METHODS  ...  47  

Protocols  ...  47  

Fly  strains  ...  47  

Fixation  and  fluorescence  immunostaining  of  Drosophila  embryos  ...  47  

RNA  in  situ  hybridization  of  Drosophila  embryos  ...  48  

Materials  ...  51  

In  situ  hybridization  in  embryos  and  dot-­‐blot  ...  51  

Fluorescent  embryo  immunostaining  ...  52  

Tables  ...  53  

Methods  ...  55  

Building  the  pYex8abdAGFP  plasmid  ...  55  

Building  the  pYex8UAShsp70ex8GFP  plasmid  ...  56  

Annotation  of  the  ELAV-­‐binding  coordinates  in  the  iab-­‐8  ncRNA  transcriptional  unit  .  56   RESULTS  ...  57  

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Chapter  I:  CNS-­‐specific  transcription  of  the  iab-­‐8  ncRNA  ...  57  

The  iab-­‐8  ncRNA  transcript  invades  the  abd-­‐A  locus  in  a  tissue-­‐specific  manner  ...  57  

Chapter  II:  Study  of  the  regulation  of  abdominal-­‐A  by  the  iab-­‐8  ncRNA    in  CRISPR-­‐ mediated  mutations  of  the  BX-­‐C  ...  65  

The  iab-­‐8  ncRNA  terminates  before  reaching  the  abd-­‐A  locus  in  the  CRISPR-­‐mediated   inversion  of  the  BX-­‐C  ...  67  

The  large  CRISPR-­‐mediated  deletions  affect  the  termination  of  the  iab-­‐8  ncRNA  as  well   as  the  regulation  of  its  promoter  ...  68  

Chapter  III:  Repression  of  an  exogenous  downstream  gene    by  the  iab-­‐8  ncRNA  ....  72  

Proving  the  ability  of  the  iab-­‐8  ncRNA  to  repress  downstream  genes  ...  73  

The  abdA:GFP  transgenic  construct  reproduces  the  expression  of  abd-­‐A,  and  is  partially   repressed  in  PS13  of  the  embryonic  central  nervous  system  ...  74  

The  abdA:GFP  construct  is  repressed  by  the  expression  of  the  iab-­‐8  ncRNA  in  a  cis-­‐ dependent  manner  ...  76  

The  insertion  of  the  abdA:GFP  construct  between  the  iab-­‐8  ncRNA  locus  and  the  abd-­‐A   locus  has  no  effect  in  the  interaction  between  the  iab-­‐8  ncRNA  and  abd-­‐A  ...  79  

The  abdA:GFP  transgene  cannot  exert  transcriptional  interference  over  abd-­‐A  ...  80  

Chapter  IV:    Attempt  to  force  ectopic  repression  of    abdominal-­‐A  by  transcriptional   interference  ...  83  

Reproducing  the  repression  of  abd-­‐A  by  ectopic  expression  of  the  terminal  exon  of  the   iab-­‐8  ncRNA  ...  83  

The  UAShsp70ex8GFP  construct  is  able  to  force  the  expression  of  the  exon  8  of  the  iab-­‐8   ncRNA  in  areas  where  it  is  not  usually  expressed,  but  it  is  unable  to  repress  abd-­‐A  by   transcriptional  interference  ...  84  

The  GAL4-­‐driven  expression  of  the  UAShsp70ex8GFP  transgene  is  reduced  in  PS13  and   PS14  of  the  embryonic  central  nervous  system,  due  to  the  expression  of  the  iab-­‐8   ncRNA  ...  86  

Chapter  V:    The  role  of  ELAV  in  transcriptional  interference  ...  90  

The  pan-­‐neuronal  gene  ELAV  ...  90  

The  terminal  exon  of  the  iab-­‐8  ncRNA  is  enriched  in  potential  ELAV-­‐binding  sites,  and   ELAV  is  detected  bound  to  its  genomic  area.  ...  93  

ELAV  is  not  necessary  for  the  expression  of  the  iab-­‐8  ncRNA  in  the  embryonic  CNS,  but   the  production  of  the  neural-­‐specific  iab8/abdA  isoform  of  this  gene  is  ELAV-­‐ dependent  ...  96  

The  iab-­‐8  ncRNA  acts  as  a  repressor  of  abd-­‐A  expression  only  in  neurons  ...  99  

Effect  of  the  loss  of  function  of  elav  in  the  expression  pattern  of  abdominal-­‐A  ...  101  

ELAV  mediates  transcriptional  interference  of  the  abdA:GFP  transgene  ...  103  

Chapter  VI:  Possible  evolutionary  conservation  of  transcriptional  interference  as  a   repressive  mechanism  ...  107  

The  readthrough  transcription  of  the  iab-­‐8  ncRNA  into  abd-­‐A  is  a  conserved  feature  of   this  gene  ...  107  

DISCUSSION  AND  CONCLUSIONS  ...  111  

Implications  ...  111  

Intergenic  splicing  in  Hox  clusters  ...  111  

Transcriptional  interference  and  genomes:  the  TIN  hypothesis  ...  113  

Possible  origin  of  the  iab-­‐8  ncRNA  ...  114  

The  enhancer  hypothesis  ...  114  

The  "lost  Hox"  hypothesis  ...  115  

The  iab8/abdA  chimeric  RNA:  A  new  gene  in  the  BX-­‐C?  ...  117  

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The  abd-­‐Ar  isoform  hypothesis  ...  117  

abd-­‐A  repression  in  the  posterior  CNS  ...  119  

ABD-­‐A  as  a  neuropeptidergic  determinant  ...  119  

Conclusions  ...  121  

The  iab-­‐8  ncRNA  exerts  transcriptional  interference  over  abd-­‐A  ...  121  

The  splicing  of  the  iab-­‐8  ncRNA  may  be  necessary  for  transcriptional  interference  ..  124  

ELAV  is  necessary  for  transcriptional  interference  ...  126  

Conservation  of  transcriptional  interference  ...  127  

Future  perspectives  ...  128  

Ectopic  ELAV-­‐mediated  abd-­‐A  repression  ...  128  

Chromatin  studies  ...  129  

ABBREVIATIONS  ...  130  

BIBLIOGRAPHY  ...  131  

APPENDIX  (Supplementary  tables  and  figures)  ...  149  

Supplementary  tables  ...  149  

Supplementary  figures  ...  151  

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ACKNOWLEDGEMENTS

This thesis has been the product of the collaboration, since 2012 (time of Mahesh’s first publication on the repression of abd-A) between many people. Therefore, I have to thank in first place François Karch, Rob Maeda, Daniel Pauli and Fabienne Cléard, for assisting in (and sometimes performing) experiments that have been vital for the development of this work.

Said that, I would like to thank my thesis director, boss and teacher for the past 8 years, François. He gave me the opportunity, 8 years ago, to join his lab, where I have stayed as undergraduate, Master student, and PhD student. In this place I learnt the true meaning of working in science (with its lights and shadows), I learnt about Drosophila, about the BX-C, and, ultimately, I learnt about myself. For all of that, thank you, as well as for your patience with me and your efforts in teaching me to be a better scientist.

Thanks to Rob, my “unofficial thesis co-director”. A lot of time has passed since we had that first conversation, back in the summer of 2011, when I came to the lab for the first time to do the interview for my short stay. During this time you have been an excellent teacher and a good friend, and I will always be grateful for the time (and again, patience), you spent with me.

To Fabienne, thank you for all your advice and support, both in the professional and in the personal aspects during these years.

Thank you to the lab members that stayed until the last moments of my Ph.D. (Annick, Clement, Yohan and Daniel). This time would not have been the same thing in other group,

To all the past lab members, but specially Dragan and Elodie, for all the good moments we spent together inside and outside the lab. A special thank also to Bastien, my first

“student” and desk neighbor.

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Thank you to Eva, Benjamin and Jorge for their excellent technical assistance.

To Emi Nagoshi’s lab, our friends and neighbors, for all the shared moments in the corridors and the coffee room.

I have to thank also my 1st TAC committee (especially Aurelien Roux) for their support in a difficult moment of my PhD, and my 2nd TAC committee (Emi Nagoshi and Maria Gambetta), for their good advice.

In a more official tone, thank you to all those who provided reactives or mutant strains (G.Technau, C. Klämbt, C. Immarigeon and J. Benito-Sipos). And thank you also to E. Rodriguez-Carballo, M. Soller, W. Bender and A. Gould for good experiment suggestions.

To the responsibles of the Bioimaging platform (J. Bosset and C. Bauer), where I spent more than a few hours at the confocal microscope, for their excellent technical assistance.

Thank you to all my friends in Geneva, or that left already, especially Dr. Kozlov, with whom I spent hours of beer consumption and interesting conversations ranging from politics, history or science.

In Madrid, I have a special place for J. Benito-Sipos (Jony), teacher, colleague and friend.

You opened for me the doors to the fly science world and without you, this long trip would not have been possible.

And here I have to thank all my friends in Madrid:

First of all, thanks to Manu, my oldest friend. You have witnessed my evolution since primary school, and have walked this long path with me.

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To Jasmina, Josune, Hugo, Yolanda, the friends I left behind in Madrid, but that I have carried with me in my heart during all these years.

To Moisés… what to say? You are on your own hard path to become Dr. Hassan, and I know you’ll make it. Thank you for all these years of friendship and support.

Thanks to Joaquin (very soon Dr. Navajas), colleague and friend. Hope that Switzerland will be as welcoming to you as it has been to me.

To Milicern Telcontar, Maese Fängorn and Gildom Dægaladh, my brothers in Tolkien.

Finally… To my brothers in arms, the heroes without name (you know who you are). I really did this thesis 4 the Lulz.

I have reserved the last, and more important place, to my family, for all of their support.

Una mención especial para Rori, por sus buenos consejos, ánimos y apoyo en estos años.

Tenías razón, el doctorado no ha sido un camino fácil, pero ha merecido la pena (aunque no me hayan dado birrete ni toga…)

A mi abuela (y, aunque ya no esté, mi abuelo). Gracias a vosotros acabé en esta ciudad, que tanto me ha dado.

Otro lugar especial para mi tía Beatriz, mi querida madrina que tanto me ha ayudado en todos estos años (y los que les precedieron). Mi estancia en Ginebra (y luego Gaillard) no habría sido lo mismo sin ti.

Finalmente, al resto de mis tíos y primos, a los que, a pesar de la lejanía, siempre he tenido presentes.

A toda mi familia de Venezuela, a la que espero pueda ir a ver pronto.

Merci beaucoup aussi à ma récemment acquise belle-famille, pour leur soutien pendant ces temps intenses et difficiles de la fin de thése.

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Y finalmente a mis padres y mi hermano, que siempre han estado a mi lado, y que tanto me han ayudado. Estos años han sido difíciles, pero en muchos aspectos también los mejores de mi vida, y me alegro muchísimo de haber podido compartirlos con vosotros.

Este doctorado es también vuestro.

And last, but not least, to my beautiful wife Marie (I switch to English, in the end, we met speaking in that language…) You have given me the best present that a man can dream of, a beautiful baby whose smile kept me going even in the most difficult moments of these last months. Thank you for your support and patience, especially during the writing phase of the thesis… I know it has not been easy for me, and I cannot imagine how it has been for you to exert as companion of a writing PhD student and mother at the same time. For all of that, and for all of the good moments we have shared during these nearly four years, thank you.

And finally, thanks to Bastien, my dear son. You are my greatest accomplishment of this year by far. I only regret I could not spend more time with you during your first months, but I am happy that we are making up for lost time. I hope that one day you will read this, and you will be as proud of your father as I am of you.

Javier Castro Alvarez, Ph.D.

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FIGURE INDEX

Figure 1 ABD-A protein and abd-A transcript present different patterns in the embryonic CNS ... 57   Figure 2 The transcription of the abd-A locus detected in PS13 and PS14 disappears in

Fab-864 embryos ... 60   Figure 3 The transcription of the abd-A locus detected in PS13 and PS14 is CNS-specific

... 61   Figure 4 The iab-8 ncRNA presents a CNS-specific transcriptional readthrough ... 62   Figure 5 The iab-8 ncRNA has alternative isoforms ... 63   Figure 6 Five isoforms of the iab-8 ncRNA result from intergenic splicing between iab-8

and abd-A ... 64   Figure 7 CRISPR-mediated recombination allowed for the production of four different

recombinant lines, lacking different elements of the iab-8 ncRNA. ... 65   Figure 8 The CRISPR-generated mutants for the iab-8 ncRNA present different degrees

of ABD-A de-repression ... 66   Figure 9 The iab-8 ncRNA terminates prematurely in the CRISPR-mediated inversion of

the iab-8 ncRNA transcriptional unit ... 67   Figure 10 The CRISPR-mediated modifications of the BX-C affect the normal

transcription in the abd-A region ... 68   Figure 11 Deletions of part of the sequence of the iab-8 ncRNA affect its normal

termination and increase the amount of readthrough transcription into the abd-A locus ... 69   Figure 12 The cell-specific enhancers present in iab-2/iab-3 interact with the iab-8

ncRNA promoter in the del(ex8+) and del(ex8-) mutants ... 71   Figure 13 Schematic representation of the elements included in the abdA:GFP transgenic

line ... 74   Figure 14 The abdA:GFP reporter is not repressed by ABD-B in PS13 of the epidermis 74   Figure 15 The abdA:GFP reporter is repressed in PS13 of the CNS ... 75   Figure 16 The repression of the abdA:GFP gene in the CNS of PS13 disappears when the iab-8 ncRNA is not expressed upstream from it ... 77   Figure 17 Cis- vs. trans- test of the repression of abdA:GFP mediated by the iab-8

ncRNA ... 78   Figure 18 The insertion of the abdA:GFP gene upstream from abd-A does not prevent

transcriptional interference ... 80   Figure 19 The presence of the abdA:GFP transgene upstream from abd-A is not able to

rescue the de-repression of abd-A in PS13 of the CNS of Fab-864 embryos. ... 82   Figure 20 Schematic representation of the elements included in the UAShsp70ex8GFP

transgenic line. ... 84   Figure 21 The UAShsp70ex8GFP construct is able to drive ectopic transcription of exon

8 of the iab-8 ncRNA in combination with the GAL-4 transcription factor ... 85   Figure 22 abd-A does not show ectopic repression in the en-Gal4;UAShsp70ex8GFP ... 86   Figure 23 The UAShsp70ex8GFP construct is downregulated in the CNS of PS13-14 ... 87   Figure 24 The iab-8 ncRNA interferes with the interaction between GAL-4 and its target

UAS sequences ... 88  

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Figure 25 Representation of the alternative splicing of the terminal exon of the gene

Neuroglian. ... 91  

Figure 26 The iab-8 ncRNA as a potential ELAV target ... 94  

Figure 27 ELAV can be detected by ChIP binding to exon 8 of the iab-8 ncRNA ... 95  

Figure 28 The readthrough transcription of the iab-8 ncRNA is strongly reduced in elav mutants ... 97  

Figure 29 elav loss-of-function does not affect the overall levels of transcription of the iab-8 ncRNA ... 98  

Figure 30 abd-A is not expressed in embryonic neuroblasts or GMCs ... 100  

Figure 31 ABD-A is exclusively expressed in the neurons of the embryonic CNS ... 100  

Figure 32 abd-A presents diverse degrees of de-repression in PS13 of the CNS in elav embryos ... 101  

Figure 33 Design of the experimental and control crosses for the study of abdA:GFP expression in elav mutant embryos ... 104  

Figure 34 The repression by transcriptional interference of the abdA:GFP transgene is abolished in elav embryos ... 105  

Figure 35 The de-repression of abdA:GFP in elav background is similar to the one caused by the absence of transcriptional interference in Fab-864,abdA:GFP embryos ... 106  

Figure 36 abd-A expression in embryos of Drosophila virilis ... 108  

Figure 37 abd-A expression in embryos of Drosophila melanogaster ... 109  

Figure 38 Expression of the iab-8 ncRNA in embryos of Drosophila virilis ... 110  

Figure 39 Structure of the two isoforms of Abd-B generated by transcription from alternative promoters ... 111  

Figure 40 Structure of the Antp gene and its two alternative isoforms ... 112  

Figure 41 Five isoforms of the iab-8 ncRNA result from intergenic splicing between iab- 8 and abd-A ... 117  

Figure 42 Schematic representation of the genetic interactions happening between the iab-8 ncRNA, abdA:GFP, UAShsp70ex8GFP and abd-A ... 123  

Figure 43 Involvement of ELAV in the generation of the iab8/abdA chimeric RNA .... 125  

Figure 44 Representation of the alternative splicing of the terminal exon of the gene Neuroglian. Modified from (Koushika et al. 1996). ... 126  

Figure 45 The UAShsp70ex7-8GFP construct ... 128  

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RÉSUMÉ

Le complexe bithorax (BX-C) de la drosophile code pour 3 gènes Hox, Ubx, abd-A et Abd-B, responsables de l’identité des structures qui forment la partie postérieure du thorax et de tous les segments abdominaux de la mouche. Le gène abd-A est exprimé du deuxième au huitième segments abdominaux, domaine qui correspond chez l’embryon au parasegment 7 (PS7) à la partie antérieure du parasegment 13 (PS13). Conformément au principe de prévalence postérieure qui est commun aux gènes Hox, l’expression d’abd-A dans la partie postérieure du PS13 et dans le PS14 est réprimée par l'expression du gène Abd-B. Ce principe de prévalence postérieure est cependant caduc dans le système nerveux central (SNC) où les 2 protéines sont co-exprimées dans certains neurones au niveau des PS10 à 12. Néanmoins, dans le PS13, abd-A est maintenu réprimé. C’est un ARN non codant appelé l’iab-8 ncARN qui assure cette répression à la place d’Abd-B.

L’unité de transcription de l’iab-8 ncARN est longue de 92 kb, occupant toute la partie intergénique entre abd-A et Abd-B. Elle contient la matrice du microARN « mir iab-8 ».

Le promoteur de l’iab-8 ncARN se situe juste en aval de l’homéobox d’Abd-B et son extrémité 3’, 1kb en amont du promoteur d’abd-A. L’iab-8 ncARN est exprimé très tôt au cours du développement dans l’ectoderme des PS13 et 14. A partir des stades précoces de la neurogenèse, l’expression de l’iab-8 ncARN est restreinte au système nerveux central (SNC) embryonnaire, et se maintient ensuite au cours des stades larvaires et adultes. Notre laboratoire a démontré que l’iab-8 ncARN se comportait comme un répresseur très efficace de l'expression d'abd-A dans le PS13 du SNC embryonnaire, via deux mécanismes indépendants. L'un d'eux résulte de la synthèse du microARN miRiab- 8 qui cible le 3'UTR de l’ARN messager d’abd-A et empêche sa traduction. Le second mécanisme agit en cis- et dépend de l’arrivée de la transcription de l’iab-8 ncARN à proximité du promoteur d’abd-A. Ce deuxième mécanisme pourrait résulter d’une interférence transcriptionnelle.

Une série de délétions internes à l'unité de transcription de l’iab-8 ncARN induites dans notre laboratoire par CRISPR/Cas9 ont montré que la majeure partie de ses séquences pouvaient être supprimées sans affecter la répression d’abd-A par le mécanisme secondaire. Toutefois, ces délétions pouvaient potentiellement également affecter des

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éléments régulateurs assurant l’activation d’abd-A, il convenait donc d’acquérir des données supplémentaires avant de tirer des conclusions définitives.

Au cours de cette thèse, je me suis intéressé à la capacité de l’iab-8 ncARN à réprimer d'autres gènes placés en aval de son unité de transcription. Je me suis également attaché à développer un système pour forcer l’expression du dernier exon de l’iab-8 ncARN (l’exon 8) afin d’induire une interférence transcriptionnelle d’abd-A de manière ectopique.

J’ai inséré une séquence codante pour la GFP pilotée par le promoteur d’abd-A juste en aval de l’exon 8 terminal de l’iab-8 ncARN. Ce gène rapporteur reproduit exactement l’espace intergénique entre la fin de l’iab-8 ncARN et le promoteur d’abd-A. Comme il ne contient pas de séquences cibles pour miRiab-8, ce gène rapporteur GFP me permet d’évaluer la force de la répression due à l’interférence transcriptionnelle en m’affranchissant de l’activité de miRiab-8. Comme anticipé, j’observe l’expression de la GFP qui reproduit exactement le patron d’expression endogène d’abd-A du PS7 au PS12.

J’observe par contre une légère dé-répression de la GFP au niveau du PS13. La différence d’expression de la GFP entre les PS12 et PS13 résulte donc de l’interférence transcriptionelle exercée par l’iab-8 ncARN sur le promoteur abd-A du gère GFP rapporteur. Aidé des membres de mon laboratoire, nous avons recombiné la délétion du promoteur de l’iab-8 ncARN (la mutation Fab-864) sur le chromosome portant le transgène abd-A-GFP. Nous avons alors assisté avec satisfaction à la dé-répression compète de la GFP dans le PS13, qui atteint un niveau d’expression similaire à celui du PS12. J’ai également pû démontrer la nature cis- du mécanisme répresseur, en constatant que la GFP était toujours sous-exprimée dans le PS13 lorsque la mutation Fab-864 se trouve en trans- par rapport au gène rapporteur GFP. Finalement j’ai observé que le gène rapporteur lui même n’est pas capable d’exercer de l’interférence transcriptionnelle sur le promoteur abd-A directement en aval. Par contre, l’insertion du gène rapporteur GFP entre le dernier exon de l’iab-8ncARN et le promoteur du gène abd-A endogène ne perturbe pas l'interférence transcriptionnelle par rapport à ce dernier, suggérant que l'interférence transcriptionnelle exercée par l’iab-8 ncARN est un mécanisme extrêmement résilient. Afin de tester cette conclusion, j’ai décidé de créer un chromosome qui me permette de contrôler la transcription de l’exon 8 à l’aide du système Gal4::UAS. A cet effet, j’ai inséré un promoteur UAS-hsp70 juste en amont de l’exon 8.

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L'expression ectopique de cette construction à l'aide de différents pilotes Gal-4 n'a toutefois pas été en mesure de provoquer une répression ectopique d’abd-A. Par contre cette expérience m'a permis de prouver l'efficacité de l’interférence transcriptionnelle exercée par l’iab-8ncARN qui lui est capable d’empêcher la liaison de Gal4 aux séquences UAS en amont de l’exon 8.

Toutes ces données suggèrent que la capacité de l’iab-8 ncARN à exercer de l’interférence transcriptionnelle sur un quelconque promoteur en aval soit limitée au SNC.

Dans l’espoir d’identifier des facteurs capables responsables de cette spécificité, j’ai examiné les séquences des exons de l’iab-8 ncARN, et j’ai trouvé des séquences de liaison au niveau de l’exon 8 pour la protéine Elav. Cette observation est très intriguante car Elav est connue pour être un facteur de liaison à l’ARN qui est exprimé spécifiquement dans le SNC. De plus des données publiées de Chipseq à l’échelle génomique confirment un enrichissement d’Elav au niveau de l’exon 8. Par hybridation in situ sur des embryons mutants pour elav, j’ai pu observer une dé-répression partielle d’abd-A dans le PS13 au niveau du SNC qui pourrait être interprété comme un échec de l’interférence transcriptionnelle. Je l'ai confirmé en montrant que la perte de fonction d'elav provoque une dé-répression totale du transgène abdA: GFP dans le PS13 du SNC.

Lors de ces expériences, j'ai également mis en évidence l’existence d’une fusion entre l’iab-8 ncARN et l’unité de transcription d’abd-A qui apparait spécifiquement dans le SNC. Ce transcrit de fusion est dépourvu de l’exon 8 terminal de l’iab-8 ncARN et des premiers exons d’abd-A et n’a pas de capacité codante. Mes observations m’amènent à penser que ce transcrit de fusion est la conséquence du mécanisme d’interférence transcriptionelle. La genèse de ce transcrit de fusion est conservée au cours de l’évolution car j’ai également pu le mettre en évidence chez D. virilis, une des espèces de drosophile parmi les plus éloignées de la branche qui a donné naissance à D.melanogaster.

Les expériences décrites dans ma thèse confirment donc que l’interférence transcriptionnelle exercée par l’iab-8 ncARN sur abd-A est un mécanisme efficace pour maintenir une prévalence postérieure dans le SNC, tout en autorisant la co-expression d’abd-A et Abd-B dans certaines populations de neurones de parasegments plus antérieurs,

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où l’iab-8 ncARN n’est pas exprimé. Ce mécanisme est conservé au cours de l’évolution.

L’existence de la fusion transcriptionnelle entre l’iab-8 ncARN et abd-A est réminiscente de l’organisation de l’unité de transcription qui code pour les isoformes « m » et « r » d’Abd-B dans les PS13 et PS14. L’existence de produits de fusions entre les unités de transcription Ubx et Antp chez certaines espèces de crustacés supporte notre idée que l’interférence transcriptionnelle ait pu être un mécanisme à la base du phénomène de la prévalence postérieure. Par ailleurs de nombreuses nouvelles données suggèrent que la production de transcrits chimériques n’est pas un phénomène si rare et certains auteurs suggèrent que de tels mécanismes ont pu être instrumentaux pour la génération de nouveaux produits protéiques.

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ABSTRACT

The Bithorax Complex of Drosophila melanogaster is a gene cluster that comprises 3 Hox genes: Ubx, abd-A and Abd-B, which together are responsible for the generation of the structures forming the posterior thorax and all the abdominal segments of the fly. The gene abd-A is expressed from the 2nd abdominal segment (PS7 in the embryo) to the 8th abdominal segment (anterior PS13). In the posterior PS13 and in PS14, the expression of abd-A is prevented by the expression of the gene Abd-B, complying to the common principle of posterior prevalence characteristic of Hox genes. In the embryonic central nervous system, however, the phenomenon of posterior prevalence does not take place, as there is co-expression of ABD-A and ABD-B in certain neurons of the parasegments comprised between PS10 and PS12. abd-A is nevertheless repressed in PS13, due to the action of a noncoding transcript, the iab-8 ncRNA.

The iab-8 ncRNA is a 92Kb long transcript transcribed in a distal-to-proximal direction (from Abd-B to abd-A), having its transcriptional end only 1Kb upstream from the promoter of abd-A. It is the template of a microRNA hairpin that is processed, generating the mir-iab-8 microRNA. The iab-8 ncRNA is expressed in PS13 and PS14 of the embryonic ectoderm, and after neural differentiation its expression becomes restricted to PS13-14 of the embryonic, larval and adult CNS. Previous studies made in our laboratory showed that the iab-8 ncRNA behaves as a very efficient repressor of abd-A expression in the embryonic CNS, via two independent mechanisms. One of them is the generation of the mir-iab-8 microRNA, which targets the 3'UTR of abd-A and prevents translation in a surprisingly efficient manner. The second mechanism is only able to act in cis-, and depends on the transcription of the iab-8 ncRNA reaching the promoter of abd-A. Due to these facts, it was proposed that this second mechanism was transcriptional interference.

A series of CRISPR-mediated modifications of the transcriptional unit of the iab-8 ncRNA made in our laboratory showed that most of its sequence could be deleted without affecting the repression of abd-A by this secondary mechanism. However, these modifications could potentially modify the regulatory elements that mediate abd-A

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expression, and caution should be exerted when considering the conclusions derived from their study.

I set as the objectives of this thesis to provide additional and solid evidence to confirm the hypothesis of transcriptional interference. I was interested in studying the potential capacity of the iab-8 ncRNA to repress other genes placed downstream from its transcriptional unit. In addition, I was interested in the potential capacity of the iab-8 ncRNA to exert transcriptional interference outside of PS13 of the CNS, by ectopically forcing the transcription of its terminal exon.

During my studies I could characterize a transcriptional readthrough generated by the iab-8 ncRNA, which skips the terminal exon of the iab-8 ncRNA and splices into the transcriptional unit of abd-A. It generates a chimeric RNA that lacks the 5'exons of abd-A and therefore, does not produce ABD-A protein. This transcript seems to be CNS-specific, which led me to propose that it might be related to the transcriptional interference mechanism acting over abd-A.

A GFP gene driven by the promoter of abd-A and inserted between the terminal exon of the iab-8 ncRNA and the TSS of the endogenous abd-A copy shows a similar expression pattern as abd-A. It is slightly de-repressed in PS13 of the CNS, due to the absence in the 3'UTR of this transgene of target sites for the mir-iab-8 microRNA. However, the expression of the abdA:GFP construct in PS13 of the CNS does not reach the levels observed in PS12 of the same tissue, indicating that there is a secondary repressive mechanism acting over it. I studied the effect of recombining the abdA:GFP transgene into a Fab-864 chromosome, which carries a deletion of the promoter of the iab-8 ncRNA.

In absence of upstream transcription from the iab-8 promoter, abdA:GFP is completely de-repressed in PS13 of the CNS. I could also test for the cis- nature of this repression, as the Fab-864/abdA:GFP trans-heterozygous embryos still show GFP repression in this parasegment. Interestingly, the abdA:GFP transgene itself is not able to repress expression of the endogenous abd-A, despite being expressed upstream from the abd-A promoter. The insertion of this construct between the iab-8 ncRNA last exon and abd-A

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does not perturb transcriptional interference over abd-A. These facts suggest that transcriptional interference is an extremely resilient mechanism, but that it can only be exerted by the iab-8 ncRNA.

I tested this last statement by building a modified exon 8 construct (UAShsp70ex8GFP) whose expression could be ectopically driven using the GAL4/UAS system. The ectopic expression of this construct using different Gal-4 drivers, however, was unable to cause ectopic repression of abd-A. This construct allowed me to prove the extreme strength of transcriptional interference caused by the endogenous iab-8 ncRNA transcript, as it was able to prevent effective GAL-4 binding to its UAS sequences in PS13-14 of the embryonic CNS.

The data obtained by the study of the abdA:GFP and of the UAShsp70ex8GFP constructs suggested that the generation of the CNS-specific readthrough of the iab-8 ncRNA was important for the occurrence of transcriptional interference over abd-A. I tried to look for potential splicing factors involved in this process. The analysis of the sequence of exon 8 of the iab-8 ncRNA suggested the involvement of the neural-specific RNA binding protein ELAV in this process. This was reinforced by the analysis of published data involving genome-wide ChIPseq against ELAV, that could detect ELAV binding around exon 8 genomic area. Using in situ hybridization on elav embryos, I could monitor the absence of transcriptional readthrough of the iab-8 ncRNA in these mutants. This was a specific effect, as the overall expression level of the iab-8 ncRNA was not affected by the elav mutation. I showed that this correlated with a partial de-repression of abd-A in PS13 of the CNS, which could be interpreted as a failure of transcriptional interference. I confirmed this by showing that the loss-of-function of elav caused a total de-repression of the abdA:GFP transgene in PS13 of the CNS.

Finally, I have shown that the generation of the iab-8 ncRNA readthrough and its invasion of the transcriptional unit of abd-A is an evolutionary conserved phenomenon, which also takes place in the BX-C of the basal Drosophila species D. virilis. This is an

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interesting fact, taking into account that similar readthrough transcription events have been detected in the Hox clusters in very different species.

This thesis has further confirmed transcriptional interference as a potent repressive mechanism by which the iab-8 ncRNA prevents expression of abd-A in PS13 in the embryonic CNS. At the same time the iab-8 ncRNA allows the breaking of the posterior prevalence between abd-A and Abd-B in anterior parasegments of the CNS, bringing up the possibility of ABD-A and ABD-B being co-expressed in some neurons, which might be important for the generation of specific neuron populations.

The generation of the iab8/abdA chimeric RNA might also be an interesting novelty in the field of Hox genes. It has been shown that the production of chimeric transcripts might not be as rare as it was initially thought, and some authors have suggested that they might be important for gene regulation, as well as for the generation of new protein products.

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INTRODUCTION

Hox genes

The positioning of body structures along the anterior-posterior axis of bilaterian organisms is determined by the Hox genes, which constitute a subset of a much larger family, the homeobox genes. These genes code for transcription factors containing a helix-turn-helix DNA-binding domain called the homeodomain (McGinnis et al. 1984, Scott and Weiner 1984, Gehring and Hiromi 1986, Kornberg 1993). Homeobox genes have been found in all eukaryotes, and recent work has suggested that this particular DNA-binding domain dates back to the archaeal genome (Bozorgmehr 2018).

Hox genes, or "homeotic genes", receive their name from the phenotypes caused by their misexpression, which already constitute a good indicator of their function. The term

"homeotic transformation" was first used by William Bateson to describe in plants the transformation of one structure into another (Bateson 1894). These transformations were later observed in animals, and thoroughly described in Drosophila thanks to the work of Edward Lewis, who mapped the genetic positions of the known alleles that caused these phenotypes, proposing that a transformation that affected to a body segment corresponded to the abnormal expression of a given Hox gene in this segment. Therefore, by deduction, the function of the Hox genes was to, due to their differential expression along the A-P axis, provide identity to each segment (Lewis 1978).

Hox genes appear associated in genomic clusters (usually denominated "homeotic complexes"). This surely reflects the diversification of the Hox genes from an ancestral gene by recurrent duplication events (Lewis 1978, Gehring et al. 2009). Clustering is a common feature, in both prokaryotes an eukaryotes, between genes that need to be regulated in a coordinated manner, from the lactose operon of Escherichia coli (Vilar et al. 2003) to the human globin gene cluster (Proudfoot et al. 1980).

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The number of genes in the Hox Complex (and the number of clusters) is variable between species, as through evolution, the Hox Complex has suffered from a number of rearrangements, ranging from duplications into other chromosomes to (as it is the case of Drosophila) disintegration, generating two homeotic clusters in the same chromosome.

Drosophila melanogaster presents two clusters of Hox genes: the Antennapedia Complex (ANTP-C) and the Bithorax Complex (BX-C). These two complexes arose from the split of an ancestral cluster between the Hox genes Antennapedia and Ultrabithorax. Other species of Drosophila present breakings of the Homeotic Complex at different locations (Von Allmen et al. 1996, Lewis et al. 2003), which has led several authors to suggest that, in these animals, there is not a strong evolutionary pressure to keep the clusters together.

For extensive reviews on this topic refer to (Ferrier and Minguillon 2003, Garcia- Fernandez 2005, Gehring et al. 2009).

One intriguing characteristic of Hox gene expression along the A-P axis is that it follows its position along the cluster, as Lewis had first described. This means that the order of expression of Hox genes along the A-P axis follows the same order in which they are located relatively to one another in the chromosome. This phenomenon, that we know today as colinearity, is a conserved feature of Hox genes through evolution (Gaunt 1988).

In vertebrates, colinearity is not only spatial, but also clearly temporal, that is, Hox genes are activated sequentially in time during the embryonic development (Gaunt and Strachan 1996). Indeed, it has been proposed that colinearity reflects the temporal activation of Hox genes during the segmentation of the embryo, which occurs in a gradual manner, being the anterior segments the first to be formed. In the case of Drosophila, where all segments are formed at the same time, clustering is not so strictly needed, as temporal colinearity has also been lost (Duboule 1992, Von Allmen et al. 1996).

Finally, and closely related to colinearity, Hox genes present the characteristic phenomenon of posterior prevalence. This means that posterior Hox genes suppress the activity of anterior ones (Gonzalez-Reyes et al. 1990, Bachiller et al. 1994). In most cases, a posterior Hox gene represses the expression of the anterior genes in the cells where it is expressed. For example, Ultrabithorax, the most anterior gene of the BX-C, is

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repressed by abd-A and by Abd-B, the posterior ones (Struhl and White 1985). There are some exceptions to this rule, which will be explained in detail in the next sections.

The Bithorax Complex of Drosophila melanogaster

The Bithorax Complex (BX-C) of Drosophila melanogaster is a genomic area of 300 Kb, located in the chromosome 3R, that comprises the necessary elements for determining the correct positioning along the anterior-posterior axis of the structures that give rise to the posterior thoracic and abdominal region of the adult fly (Lewis 1978, Karch et al. 1985).

This region of the body is composed of 9 segments (one thoracic and eight abdominal), and it derives from the embryonic parasegments PS5-PS14. At this point it is convenient to clarify that, during the embryonic development of Drosophila, the embryo is divided into a parasegmental organization, where each parasegment corresponds to the posterior compartment of an adult segment and the anterior compartment of the next one (for example, PS13 corresponds to the posterior seventh abdominal segment, A7, and the anterior A8). The expression of Hox genes follows this division (Martinez-Arias and Lawrence 1985), and therefore I will refer to parasegments from now on.

Ed Lewis and the BX-C

The studies of Edward Lewis of the Bithorax Complex showed that the expression of the homeotic phenotypes along the anterior-posterior axis followed the position of the alleles that caused each one of them along the chromosome. He proposed that this was the result of the sequential activation of gene products located in the BX-C (following the principle of colinearity explained in the first section). He stated that, once a gene was activated in a given parasegment, it would remain active until the posterior end of the animal. Finally, he proposed that the activation of each gene was controlled by cis- acting elements which controlled each gene in an independent manner. Interestingly, Lewis proposed that the genes were maintained by default in a inactive state due to the repressive activity exerted by the gene Polycomb (Pc) (Lewis 1978).

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In this initial model, Lewis proposed the existence of at least nine "segment-specific functions". Later studies (Sanchez-Herrero et al. 1985, Tiong et al. 1985, Casanova et al.

1987, Martin et al. 1995) showed that, in fact, the BX-C was only composed of three Hox genes: Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B).

The Bithorax Complex Hox genes

If the BX-C only contains 3 genes, how does it control differential gene expression in 9 different parasegments? The vast amount of mutations studied inside the BX-C that showed homeotic phenotypes allowed, less than 10 years after the predictions of Ed Lewis, to propose a model on the function of the BX-C. The proposed model stated that the BX-C is divided into 9 independent regulatory domains. Each domain is responsible for the activation of a different expression pattern for a single gene from a given parasegment towards the posterior end of the embryo (Figure I). The unique combination of Hox expression patterns generated in this manner specifies the identity of each parasegment. In this way, the domain iab-2 would be responsible for assigning an expression pattern to the gene abd-A from PS7, iab-3 from PS8, etc (Peifer et al. 1987).

The expression pattern of each one of the Hox genes, and the corresponding regulatory domains, are depicted in Figure I.

Figure I. Genetic composition of the BX-C

Schematic representation of the expression pattern of the three Hox genes present in the BX-C in the embryo (A) and its correspondence with the adult body (B). The color scheme represents the regulatory domains of the BX-C that drive expression of each HOX gene, represented in B. Figure modified from (Maeda and Karch 2006).

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Ubx becomes active in PS5 of the embryo, and remains present all along the axis until PS12 (Akam et al. 1985, Hogness et al. 1985). This expression pattern is regulated by the domains abx/bx and bxd/pbx, and is essential for the determination of the identity of the third thoracic segments (T3) and the first abdominal segment (A1) of the adult fly (Casanova et al. 1985, White and Wilcox 1985, Muller and Bienz 1991).

abd-A begins to be expressed in PS7 of the embryo, remaining active until the anterior compartment of PS13, and determines the identity of the abdominal segments A2, A3 and A4 of the adult fly. The cis-regulatory sequences that confer this expression pattern are located in the domains of the BX-C iab-2, iab-3 and iab-4 (Karch et al. 1990, Macias et al. 1994).

Abd-B is expressed from the embryonic PS10, determining the identity of the last abdominal segments (A5, A6, A7, A8 and A9) of the adult. Its expression is regulated by the domains iab-5, iab-6, iab-7 and iab-8,9 (Casanova et al. 1986, Celniker et al. 1989, Celniker et al. 1990, Sanchez-Herrero 1991). Interestingly, this gene can start its transcription from different promoters, giving rise to two different protein isoforms (ABD-Bm and ABD-Br) (Casanova et al. 1986, Zavortink and Sakonju 1989).

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Regulation in the BX-C: The "open for business" model

Once established the fact that 9 regulatory domains control the differential expression of each of the 3 Hox genes of the BX-C in each of the embryonic parasegments between PS5 and PS14, how does this regulation take place? Adapting the existence of cis- regulatory domains to the model proposed by Ed Lewis that required sequential activation of each gene in a colinear manner, the prediction made in (Peifer et al. 1987) was that the domains are either "inactive" or "active" in each parasegment, depending on their chromatin state. This would mean that the regulatory regions present on each of the domains would be able to drive transcription of their target Hox gene only if they were on a parasegment where the domain was "open". Accordingly, the chromatin of the BX-C, repressed in anterior parasegments of the embryo, would be progressively "opened"

(turned to a permissive state) as we travel along the anterior-posterior axis. This is known as the "open for business" model. For a comprehensive review on the subject refer to (Maeda and Karch 2015).

This model was definitely confirmed years later, by the insertion in various areas of the BX-C of P-elements containing a lacZ gene, able to do enhancer trapping. Each one of the lines containing a P-element expressed lacZ from a different parasegment. This pattern was highly reproducible and depended on the region of the BX-C in which the P-element had landed (Figure II). For example, a line containing a P-element that landed on the region of iab-2 shows a lacZ expression pattern that begins in PS7 and remains active until the posterior end of the embryo. This is explained by the promoter of the lacZ reporter gene "trapping" the enhancers from the iab-2 region in the parasegments where this region is in an active state (Bender and Hudson 2000).

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Figure II. P element homing in the BX-C leads to restricted lacZ expression patterns Dissected embryos showing restricted lacZ expression patterns coming from P elements inserted in various locations of the BX-C. Reproduced from (Maeda and Karch 2006), the figure is an adaptation from (Bender and Hudson 2000).

One year later, a modification of this study, but based on the insertion of a lacZ gene whose expression was controlled by UAS sites instead of by enhancer trapping, showed that the GAL4 transcription factor was unable to access its target UAS sites inserted in a given regulatory domain in those parasegments where the domain was predicted to be inactive. Based on this observation, the authors proposed that the inactivation of the regulatory domains actually corresponded with a reduction in chromatin accessibility (Fitzgerald and Bender 2001).

The demonstration that the activation of the regulatory domains corresponded to the switch of a repressive chromatin mark to an active mark requires the possibility of purifying nuclei specific to single parasegments, a prerequisite that became only recently

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available through the elegant work of the Bender's laboratory (Bowman et al. 2014). In this work, Bowman et al. created a series of Gal-4/Gal80 enhancer trap lines, that when combined appropriately, generated embryos in which Gal-4 is active in specific single parasegments (Figure III,A).

This technique showed that, in different parasegments, the profile of H3K27 trimethylation (a common mark characteristic of Polycomb-repressed chromatin) in the chromatin of the BX-C was different (Figure III,B). The H3K27me3 pattern was high in the domains predicted to be inactive in each parasegment. Active domains, on the other hand, presented low H3K27me3 and high H3K27 acetylation, a typical chromatin mark of active enhancers.

This established for the first time a direct correlation between the Lewis model and the increasing/decreasing of chromatin accessibility in the domains of the BX-C along the anterior-posterior axis.

Figure III. A parasegment-specific Gal-4 system for cell sorting

A. Different lines expressing combinations of Gal4+Gal80 drive expression of a reporter gene in a single parasegment, during embryonic development.

B. H3K27me3 profile of the BX-C in nuclei extracted from whole embryos (mixed) and from single parasegments between PS4 and PS7. CTCF binding (red lines) marks the location of the boundaries between domains.

Modified from (Bowman et al. 2014).

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Cis-regulatory domains of the BX-C

The "open for business" model established in (Peifer et al. 1987) made some surprisingly accurate predictions about the elements that should compose each of the cis- regulatory domains of the BX-C:

- Each of these domains must have elements able to "sense" the position of the cell along the A-P axis and, therefore, to determine the active or inactive state of the domain itself.

- It must have cell-specific enhancers that allow for the activation of its target Hox gene in a particular manner, different in each parasegment.

- Finally, the division of the BX-C into "domains" implied the existence of clear limits between each of those regions.

The techniques available at that time had not allowed yet for a fine dissection of each of the domains, but time has proven that indeed those three predictions were inherently correct, and corresponded to discrete elements present in each of the cis-regulatory modules of the BX-C. The fact that in each of the modules we find similar kinds of elements is yet another argument in favor of the proposition made in (Lewis 1978), by which the BX-C arose from events of duplication and divergence. For comprehensive reviews on the subject, see (Maeda and Karch 2006, Maeda and Karch 2015).

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At the beginning of embryonic development, the maternal, gap, and pair-rule genes are differentially expressed along the anterior-posterior axis, creating gradients and unique combinations of gene products that divide the embryo in 14 parasegments. Each of the regulatory domains of the BX-C, in a process that is dependent on these proteins, determines the activation and expression pattern of one Hox from a given parasegment (White and Lehmann 1986, Irish et al. 1989).

But how is this positional information transmitted to each of the domains? Most of the early studies of the cis-regulatory domains were made using as approach a typical enhancer activity assay. In this assays, a piece of DNA is cloned upstream or downstream of a reporter (lac-Z at the time) with a minimal promoter. If the DNA fragment is an enhancer, it will confer a reproducible expression pattern to the lac-Z gene (Simon et al.

1990). This approach allowed for the characterization of the first tissue- and cell- specific enhancers of the BX-C, confirming the existence of these elements in the BX-C regulatory domains that had been predicted in (Peifer et al. 1987).

Surprisingly, certain sequences did not drive transcription of the lac-Z reporter in a tissue-specific manner, but in a restricted parasegment of the embryo. The authors called them "parasegmental control elements" and predicted that they might be responding to the variation in the concentration of gap or pair-rule gene products along the axis (Simon et al. 1990, Muller and Bienz 1992). Later, these sequences received the name of

"initiators". They have been identified in different regulatory domains of the BX-C, and they are indeed composed by discrete binding sequences for gap and pair-rule proteins (Qian et al. 1991, Zhang et al. 1991, Busturia and Bienz 1993, Zhou et al. 1999, Barges et al. 2000, Shimell et al. 2000).

Our laboratory has finally confirmed that the activity of the initiator only depends on the positional information along the A-P axis, and not on the regulatory domain in which it is present. In this way, the ectopic insertion of the initiator of the regulatory domain iab-5 (which activates this domain in PS10) into the iab-6 domain (normally active from PS11)

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leads to the activation of the iab-6 domain in PS10, and the subsequent transformation of PS10 into PS11 (Iampietro et al. 2010).

Boundaries

The model proposed in (Peifer et al. 1987) about the organization of the BX-C into domains implied the existence of some kind of boundaries, that delimited each of the regulatory domains to ensure its independence from the neighbor. A first hint on the existence of such elements arose with the localization of the Mcp1 deletion (Karch et al.

1985). The confirmation of this prediction came in 1990, with the discovery of Fab-7, the boundary element between the regulatory domains iab-6 and iab-7 (Gyurkovics et al.

1990), and it was rapidly followed by descriptions of boundaries between the other domains, the latest one being Fub, between bxd and iab-2 (Bender and Lucas 2013), for review see (Maeda and Karch 2015). From a molecular point of view, however, a boundary is a complex of DNA sequences able to recruit insulator proteins that protect the chromatin located on one side from the effects of enhancers and other sequences located in the other. In the BX-C, these proteins are mainly the insulator CTCF (Smith et al. 2009) and, in the case of the Fab-7 boundary, the GAGA factor (Wolle et al. 2015). In fact, the boundaries act as limits for the waves of chromatin derepression caused by the initiators. In accordance, the deletion of a boundary has as an effect the premature activation of a domain (par example, the deletion of Fab-7 causes the ectopic activation of the iab-7 domain in PS11, therefore causing a transformation of PS11 towards PS12).

This boundary activity is not parasegment specific, and due to this, a boundary of the BX-C can nearly totally substitute for the activity of another (Gyurkovics et al. 1990, Iampietro et al. 2008).

Maintenance elements

In the reporter experiments described in (Simon et al. 1990) that first proposed the existence of the initiators, it was observed that, although the lac-Z reporter was activated in the early embryo following a very restricted pattern, this restriction was lost in later embryonic stages. This is due to the loss of positional information that the gap and pair- rules initially provide at the beginning of development. However, the expression pattern

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29

of Hox genes is not lost, due to the activity of the so called "maintenance elements".

These sequences recruit proteins from the Polycomb family or the Trithorax family, which in turn maintain the active or inactive state of one Hox gene in one parasegment, which had been previously determined during the initiation phase (Simon et al. 1993, Simon 1995, Gross and McGinnis 1996, Strutt et al. 1997, Brock and van Lohuizen 2001, Schuettengruber et al. 2017). This is done via chromatin modifications of different types, which include, but are not restricted to, histone methylation, acetylation, and nucleosome remodeling. Most of these mechanisms are reviewed in (Simon and Tamkun 2002).

Intergenic RNAs in the Bithorax Complex

Intergenic transcription is a common feature of eukaryotic genomes. Noncoding transcription has been shown to perform diverse and important functions (Amaral and Mattick 2008), and the Hox clusters are no exception. During the past years, several noncoding RNAs have been identified in Hox clusters; one of the best known is the human HOTAIR (Rinn et al. 2007), involved in Polycomb-mediated repression of several HoxD genes (Li et al. 2013).

The early domain-specific transcription

Before the Hox genes begin to be expressed, each domain of the BX-C generates transcripts in a segment-specific pattern (iab-2 in PS7, iab-3 in PS8, etc.). This segment- specific early transcription was first described in (Sanchez-Herrero and Akam 1989), and more thoroughly studied in (Bae et al. 2002). Although much is not known about these early transcripts, they seem to originate from the initiator elements present in the regulatory domains (S. Galletti, unpublished). Some authors have suggested that this transcription could intervene in the initial activation of the domains, by displacing Polycomb proteins from the chromosome (Bender and Fitzgerald 2002, Hogga and Karch 2002, Rank et al. 2002).

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30 The noncoding RNA bithoraxoid (bxd)

The bxd/pbx regulatory domain that activate Ubx expression from PS6 is also giving rise to a noncoding RNA described for the first time in (Lipshitz et al. 1987). This ncRNA is also transcribed from PS6 but with a complementary expression pattern relative to Ubx inside each parasegment. It has been proposed to repress Ubx by transcriptional interference (Petruk et al. 2006).

The iab-4 noncoding RNA

A promoter located in the iab-3 regulatory region, very close to the boundary with iab-4, is responsible for the transcription of a 6-8Kb noncoding RNA, that gets spliced and polyadenylated. This RNA was first described in 1990, where it received the name of the iab-4 ncRNA. In accordance to the location of its promoter, the iab-4 ncRNA is expressed from PS8 to PS12 (Cumberledge et al. 1990, Garaulet et al. 2014). It is the template of the mir-iab-4 microRNA (Aravin et al. 2003). This microRNA has been shown to be a repressor of Ubx, and it is analogous to mir-196, a vertebrate microRNA that targets the Hox gene HOXB8 (Yekta et al. 2004, Ronshaugen et al. 2005).

The iab-8 noncoding RNA

The transcription of the region between abd-A abd Abd-B had already been hinted by previous studies, by the detection of a distal-to-proximal transcript (Abd-B to abd-A) that could be detected by in situ hybridization in PS13-14 of the embryo using probes against this region. This transcript originates from a promoter located downstream from Abd-B, in the iab-8 regulatory domain (Sanchez-Herrero and Akam 1989, Zhou et al. 1999, Bender and Fitzgerald 2002, Rank et al. 2002).

In 2008 it became evident that this transcription corresponded to the expression of a long noncoding RNA, and that it was responsible for the generation of mir-iab-8, the complementary microRNA from the same locus as the mir-iab-4 hairpin (Bender 2008, Tyler et al. 2008).

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The iab-8 ncRNA is a 92Kb transcript, spliced and polyadenylated. Its main spliced product is composed of 8 exons, respectively derived from each of the cis-regulatory domains. It is unclear whether this splicing pattern responds to a function. Interestingly, the sequences surrounding the splice junctions delimiting each exon seem to be conserved between Drosophila species, as if it was the act of splicing that played a role.

Some minor isoforms of the iab-8 ncRNA skip exon 8, and splice into the transcriptional unit of abd-A (Gummalla et al. 2012).

Figure IV. The iab-8 ncRNA

Schematic representation of the main spliced product of the iab-8 ncRNA.

The iab-8 promoter is located in the iab-8 regulatory region, very close to the Fab-8 boundary. Therefore, it could potentially trap the enhancers that drive the expression of Abd-B in PS13. In agreement, the iab-8 ncRNA is only expressed in PS13-14 (Figure V).

It can be detected by in situ hybridization against its terminal exon already at blastoderm stage, after cellularization. This expression restricted to PS13-14 is constant throughout embryonic development, first in the ectoderm and later in the central nervous system in late embryos and larval brain (Gummalla et al. 2012, Garaulet et al. 2014, Gummalla et al. 2014). Recent observations made in our laboratory have confirmed its expression even in adult brain (Y. Frei, personal communication).

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32 Figure V. Expression pattern of the iab-8 ncRNA

In situ hybridization against the iab-6 region in embryos in blastoderm stage (A), germband extension stage (B) and germ band retraction (C), showing the expression pattern of the iab-8 ncRNA. Modified from (Gummalla et al. 2014).

The msa transcript

A transcriptome analysis throughout development identified a new lncRNA derived from the region between Abd-B and abd-A, but transcribed from a promoter located in the iab-6 region. This RNA was predominantly detected in adult males, fact from which it received the name male specific abdominal (msa) (Graveley et al. 2011).

Strikingly, both transcripts, the iab-8 ncRNA and msa, share the same splicing pattern, and they only differ by the first two exons of the iab-8 ncRNA, absent in msa. Their expression pattern, however, is completely different. While the iab-8 ncRNA is expressed from embryonic stages, msa is only expressed in the secondary cells of the accessory gland, a male-specific reproductive structure. It is the result from the transcription of an Abd-B enhancer located in the iab-6 regulatory domain of the BX-C (Gligorov et al. 2013, Maeda et al. 2018).

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A recent publication from our laboratory show that msa is also template for mir-iab-8, which apparently regulates genes in the secondary cells of the male accessory gland.

These genes are essential to elicit a proper post-mating response in females (Maeda et al.

2018).

Furthermore, recent studies performed in our laboratory have shown that msa is associated with polysomes. Indeed a short ORF located in exon 8 of the iab-8 ncRNA (exon 7 of msa) produces a micropeptide, which seems to be involved in sperm competition (C. Immarigeon, in preparation).

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