lncRNA read-through regulates the BX-C insulator Fub-1

  1. Airat Ibragimov
  2. Xin Yang Bing
  3. Yulii V Shidlovskii
  4. Michael Levine
  5. Pavel Georgiev
  6. Paul Schedl

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    This fundamental work substantially advances our understanding of how neighboring genes on a chromosome can be separately controlled in time and space. The evidence supporting the conclusions is compelling, with state-of-the-art genetic perturbations and imaging. The work will be of broad interest to geneticists and cell biologists.

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Abstract

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax ( Ubx ) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

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  1. eLife

    Author Response

    Reviewer #1 (Public Review):

    The paper reports important work in which the Fub-1 boundary of the Drosophila bithorax complex is characterized in detail. Fub-1 separates the bxd/pbx regulatory domain, which is active in PS6/A1, from the abx/bx regulatory domain, which is active in PS5/T3. The work presented provides compelling evidence that Fub-1 consists of two key elements: an insulating boundary region called HS1, which is regulated by an adjacent region called HS2. HS2 contains a promoter that is activated in PS6/A1 by enhancers in the bxd/pbx region. Read-through of HS1 by transcripts from the HS2 promoter blocks the insulating activity of HS1, allowing the bxd/pbx regulatory regions to activate Ubx transcription in PS6/A1. It has long been appreciated that boundary elements within the BX-C are regulated in a segment-specific fashion. The work presented in the Ibragimov manuscript provides a very nice example of how this segment-specific regulation can take place. For the most part, the work is very thorough and the conclusions are well-supported. However, there are a few important issues that should be addressed.

    First, throughout the manuscript, it is stated that the read-through transcription of HS1 eliminates its blocking activity. Missing, however, is a test of whether the direction of transcription of HS1 is important. That is, no construct is tested in which HS1 is inverted so that RNAs from the HS2 promoter are transcribed from the opposite strand of HS1. If read-through transcription of HS1 is all that is required to abrogate its blocking activity, such a construct should behave identically to constructs in which HS1 is not inverted. However, if the structure of the F1HS2 RNA is critical to preventing the blocking activity of HS1, inversion of HS1 relative to HS2 may render it immune to inactivation by transcripts initiated at HS2.

    This is a good point. The sequence/structure of the transcript could be important—e.g., it recruits a factor that disrupts boundary activity.

    While we didn’t do such an experiment, this scenario seems unlikely. As noted above we have replaced Fub-1 with two other BX-C boundaries Mcp and Fab-8. Their sequences are different from other and from Fub-1. Both block bxd/pbx from regulating Ubx and give an A1 LOF phenotype. To test the effects of transcription on boundary activity, we placed a P-element promoter upstream of both boundaries (so transcripts from the P-element promoter would read through boundaries towards bxd/pbx. We found that inclusion of the P-element promoter rescued the LOF phenotypes.

    Second, the terminology used to designate the constructs tested is very hard to follow and needs simplification. Since the orientation of HS1 in isolation is unimportant, perhaps just HS1 HS2, HS1 Inv(HS2), HS2 HS1, and Inv(HS2) HS1 could be used.

    We wanted to keep the terminology consistent in so far as possible with publications on other BX-C boundaries.

    Third, in many places in the manuscript genotypes are shown in which the HS1 insulator is placed into F7attP50. For these genotypes, H1 is said to block the interaction between iab-6 and iab-7, but not to support bypass activity. Readers need some help here, as they will not understand why A5 and A6 tergites are black in these genotypes, as this implies that iab-5 is able to act over the HS1 element to activate Abd-B. One explanation may be that iab-5 can promote pigmentation by acting on abd-A.

    The likely explanation is that the Fab-6 boundary is able to "bypass" the intervening HS1 insulator and target iab-5 enhancers to Abd-B promoter. There are other Fab-7 replacements in which the iab-5 enhancers are also blocked. The likely explanation is that the Fab-6 boundary is able to "bypass" the intervening HS1 insulator and target iab-5 enhancers to Abd-B promoter. We added an explanation and a review article describing to the text.

    Fourth, a more complete description of the HS1248 HS2505R genotype is needed. In this genotype, the H1 insulator is constitutively active, as H2 is inverted. Do animals of this genotype show a bxd phenotype in the larval cuticle? Do adults show a transformation of the halteres like that shown by classical bxd mutations? Answers to these questions would shed light on when H1 is active as an insulator, and whether it is active throughout PS6/A1.

    Phenotype of larval cuticle indicates a LOF transformation towards T3. We added a supplementary Figure 6-figure supplement 5 showing this. The haltere shows evidence of an LOF phenotype (Figure 6-figure supplement 6).

  2. Version published to 10.7554/elife.84711 on eLife
  3. eLife

    eLife assessment

    This fundamental work substantially advances our understanding of how neighboring genes on a chromosome can be separately controlled in time and space. The evidence supporting the conclusions is compelling, with state-of-the-art genetic perturbations and imaging. The work will be of broad interest to geneticists and cell biologists.

  4. eLife

    Reviewer #1 (Public Review):

    The paper reports important work in which the Fub-1 boundary of the Drosophila bithorax complex is characterized in detail. Fub-1 separates the bxd/pbx regulatory domain, which is active in PS6/A1, from the abx/bx regulatory domain, which is active in PS5/T3. The work presented provides compelling evidence that Fub-1 consists of two key elements: an insulating boundary region called HS1, which is regulated by an adjacent region called HS2. HS2 contains a promoter that is activated in PS6/A1 by enhancers in the bxd/pbx region. Read-through of HS1 by transcripts from the HS2 promoter blocks the insulating activity of HS1, allowing the bxd/pbx regulatory regions to activate Ubx transcription in PS6/A1. It has long been appreciated that boundary elements within the BX-C are regulated in a segment-specific fashion. The work presented in the Ibragimov manuscript provides a very nice example of how this segment-specific regulation can take place. For the most part, the work is very thorough and the conclusions are well-supported. However, there are a few important issues that should be addressed.

    First, throughout the manuscript, it is stated that the read-through transcription of HS1 eliminates its blocking activity. Missing, however, is a test of whether the direction of transcription of HS1 is important. That is, no construct is tested in which HS1 is inverted so that RNAs from the HS2 promoter are transcribed from the opposite strand of HS1. If read-through transcription of HS1 is all that is required to abrogate its blocking activity, such a construct should behave identically to constructs in which HS1 is not inverted. However, if the structure of the F1HS2 RNA is critical to preventing the blocking activity of HS1, inversion of HS1 relative to HS2 may render it immune to inactivation by transcripts initiated at HS2.

    Second, the terminology used to designate the constructs tested is very hard to follow and needs simplification. Since the orientation of HS1 in isolation is unimportant, perhaps just HS1 HS2, HS1 Inv(HS2), HS2 HS1, and Inv(HS2) HS1 could be used.

    Third, in many places in the manuscript genotypes are shown in which the HS1 insulator is placed into F7attP50. For these genotypes, H1 is said to block the interaction between iab-6 and iab-7, but not to support bypass activity. Readers need some help here, as they will not understand why A5 and A6 tergites are black in these genotypes, as this implies that iab-5 is able to act over the HS1 element to activate Abd-B. One explanation may be that iab-5 can promote pigmentation by acting on abd-A.

    Fourth, a more complete description of the HS1248 HS2505R genotype is needed. In this genotype, the H1 insulator is constitutively active, as H2 is inverted. Do animals of this genotype show a bxd phenotype in the larval cuticle? Do adults show a transformation of the halteres like that shown by classical bxd mutations? Answers to these questions would shed light on when H1 is active as an insulator, and whether it is active throughout PS6/A1.

  5. eLife

    Reviewer #2 (Public Review):

    The work presented in the manuscript addresses regulatory mechanisms in a complex genome locus, the Bithorax-Complex (BX-C) in Drosophila. Here three homeotic genes are controlled by multiple regulatory domains, each of which comprises distinct sets of cis-regulatory elements including insulators, enhancers, Polycomb responsive elements, and promoters for coding and non-coding transcripts. Despite such complexity, the authors have made good efforts to explain the context for the study and the question that they are interested in, what is the function of an evolutionarily conserved but newly defined cis-element, Fub-1?

    Fub-1 localizes at the chromatin boundary between the homeotic gene Ubx and the bxd/pbx regulatory domain, which thus predicts it is a chromatin insulator. To dissect the function of Fub-1, the authors utilized powerful and versatile gene exchange cassettes (phiC31/attp; FRT/FLP; Cre/Loxp) to engineer both the endogenous locus of Fub-1 and another insulator site Fab-7 to introduce exogenous Fub-1. Using these transgenic tools, they tested the insulator activity of Fub-1. They first confirmed that deleting Fub-1 causes changes in chromosomal configuration in the flanking region using Micro-C. However, unexpectedly, they found that Fub-1 depletion does not cause homeotic transformation, a phenotype that is expected to occur when the expression of the homeotic gene is changed due to the loss of chromatin insulators. Instead, they observed that only a sub-element within Fub-1 has an insulator function while the other sub-element that contains an active promoter suppresses insulator activity. They further demonstrated that although there is no detectable phenotype when both sub-elements are deleted, changing the direction of the promoter or stopping transcription by adding an SV40 terminator in between the two sub-elements could relieve the suppression of insulator activity. From this evidence, the authors conclude that transcriptional read-through from the active promoter of a non-coding transcript regulates the insulator activity of Fub-1.

    The finding provides a new angle to examine regulation by insulators and reveals a new function of active promoters of non-coding transcripts. The work also leaves further questions, for example, how general is such a mechanism in the genome organization of Drosophila and other organisms, and what is the significance of the mechanism given that deleting the Fub-1 insulator does not cause phenotypic outcomes in Drosophila? In the discussion, the authors elaborated on possibilities to discuss these questions.

  6. Version published to 10.1101/2022.11.13.516321v1 on bioRxiv