Research Article

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

Department of Molecular Biology, Princeton University, United States
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Russian Federation
Lewis Sigler Institute, Princeton University, United States
Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology Russian Academy of Sciences, Russian Federation
Department of Biology and General Genetics, Sechenov University, Russian Federation
Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, Russian Federation

Aug 10, 2023

https://doi.org/10.7554/eLife.84711

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Version of Record: September 12, 2023
Accepted Manuscript: August 10, 2023

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Airat Ibragimov

Xin Yang Bing

Yulii V Shidlovskii

Michael Levine

Pavel Georgiev

Paul Schedl

(2023)
lncRNA read-through regulates the BX-C insulator Fub-1

eLife 12:e84711.

https://doi.org/10.7554/eLife.84711
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Figures
Tables
Additional files

8 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements

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The organization of the genes and regulatory domains in BX-C.

The *Drosophila melanogaster* Bithorax complex (BX-C), which contains the Hox genes *Ubx*, *abd-A,* and *Abd-B*, is shown in relation to where these three genes are expressed in an embryo. There are nine *cis*…

Figure 1—figure supplement 1

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Chip-seq profiles of different chromatin proteins and histone modifications across *bxd/pbx* domain.

ChIP-seq profiles for H3K4me3, H3K27me3, Pol II, CTCF, CP190, Zw5, Pita, Elba3, Insv, Zld, and GAF (Bowman et al., 2014, p. 27; Fuda et al., 2015; Harrison et al., 2011; Ueberschär et al., 2019; Zolo…

Figure 1—figure supplement 2

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*Fub-1* sequence conservation.

Sequence comparison of *Fub-1* boundaries from *D. virilis*, *D. pseudoobscura*, *D. erecta*, *D. sechellia,* and *D. melanogaster*. The *Fub-1* sequences of *D. melanogaster* and four other *Drosophila* species were …

Figure 1—figure supplement 3

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Strategy for creating *Fub-1* deletion and replacement lines.

Top: schematic representation of the regulatory region containing the *Ubx* gene. The *F1^attP* line was obtained through the substitution of a 1168 bp region with *attP* site and the *dsRed* gene, flanked …

Figure 2

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Chromatin topology in *bxd/pbx* domain in WT and in *Fub-1* deletion.

(A) Morphology of the abdominal segments of WT and *F1^attP* flies. (B) Micro-C contact map of the WT and *F1^attP* 12–18 hr embryos at 800 bp resolution. The black arrow points to a sub-TAD formed by *Fub-…*

Figure 3

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*DsRed* and *GFP* markers trap enhancer activity in *Fub-1* replacements.

*DsRed* (in red) and *GFP* (in green) expression in stage 14 embryos in the *Fub-1* deletion and in two replacements as indicated. DAPI was used to stain nuclei (in blue). *DsRed* expression in *F1^attP-DsRed* …

Figure 4 with 2 supplements

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Testing boundary activity of *Fub-1* sequences.

(A) Top: schematic presentation of *Fab-7* substitution. Bottom: *Fub-1* fragments used in the replacement experiments. On the right side, the insulator activity with the various fragments in the *F7^attP5…*

Figure 4—figure supplement 1

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Morphology of the abdominal segments (numbered) in males carrying different variants of *Fub-1* in *Fab-7^attP50* platform in the dark field.

Figure 4—figure supplement 2

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Morphology of the abdominal segments (numbered) in males carrying *HS2⁵⁰⁵* and *HS2^505R* fragments in *Fab-7^attP50*.

Figure 5 with 1 supplement

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Read-through transcription inactivates *HS1* insulator.

(A) Schematic representation of sequences tested in *F7^attP50*. SV40 terminator is shown as a light blue arrow, and poly(A) sites are marked as vertical red lines. All other designations are the same …

Figure 5—figure supplement 1

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*Abd-B* expression in *Fab-7* replacement embryos.

The panels on the left show confocal images of embryonic CNS of *HS2^505R-HS1^248R, HS2^505R-HS1^248R-SV40,* and *HS2^505R-SV40-HS1^248R* embryos. The panels on the right show plot profiles of relative …

Figure 6 with 6 supplements

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*HS2* drives transcription in tissue-specific manner.

(A) Schematic presentation of *bxd* domain with *HS1²⁴⁸-HS2^505R* and *HS2^505R-HS1^248R* replacements. The smFISH strand-specific probes are shown as orange and light blue arrows (Supplementary file 1b and c…

Figure 6—figure supplement 1

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Expression of *bxd* and *F1^HS2* lncRNAs in NC13 WT and *F1^attP* embryos.

Stage 4 (nuclear cycle 13) WT and *F1^attP* embryos were hybridized with smFISH probes complementary to ‘sense’ strand sequences proximal to (downstream of) *Fub-1*. DAPI was used to stain nuclei (in …

Figure 6—figure supplement 2

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Expression of *bxd* and *F1^HS2* lncRNAs in stage 7/8 WT and *F1^attP* embryos.

Stage 7/8 WT and *F1^attP* embryos were hybridized to smFISH probes complementary to ‘sense’ strand sequences just proximal to (downstream of) Fub-1. DAPI was used to stain nuclei (in blue). During …

Figure 6—figure supplement 3

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Expression of *bxd* and *F1^HS2* lncRNAs in stage 11 WT and *F1^attP*embryos.

Stage 11 WT and *F1^attP* embryos were hybridized with smFISH probes complementary to ‘sense’ strand sequences just proximal to (downstream of) *Fub-1*. At this stage, segmental grooves appear. In *WT,* …

Figure 6—figure supplement 4

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Halters morphology in WT and *HS1²⁴⁸-HS2^505R* flies.

Images show a lateral view of female flies with their wings removed to visualize the halters. The segments in the dorsal cuticle are designated as follows: Al, A2, and A3. In *HS1²⁴⁸-HS2^505R* flies, …

Figure 6—figure supplement 5

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Homeotic transformation of larval segments in *HS1²⁴⁸-HS2^505R*.

(A) Ventral cuticle of WT and *HS1²⁴⁸-HS2^505R* third-instar larva. WT larvae have 11 denticle belts marking the anterior half of each thoracic and abdominal segments. These are visible as black dots. …

Figure 6—figure supplement 6

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*Ubx* expression in halter discs.

WT and mutant halters discs were probed with Ubx antibodies. All tissues are from third-third instar larvae, anterior is to the left. Ubx staining is the greatest in the central pouch. In *HS1²⁴⁸-HS2^5…*

Figure 7

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*HS2* drives transcription in a tissue-specific manner.

smFISH of stage 14 embryos of indicated genotype with a probe that targets antisense strand just distal to *Fub-1*. In WT embryos, no antisense (proximal to distal) transcripts were detected. By …

Figure 8

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Model showing changes in TAD organization during *Fub-1^HS2* transcription.

Top: In *WT,* the 3D organization of the *bxd/pbx* regulatory domain transitions from one state to another when it activates *Ubx* expression (see also Mateo et al., 2019). In PS5 (T3) and more anterior …

Tables

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Drosophila melanogaster)	Cas9	Bloomington Drosophila Stock Center	BDSC: 55821	y[1] M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2A w[1118]
Genetic reagent (D. melanogaster)	ΦC31	This lab	N/A	w[1], ΦC31 (y⁺); TM6, Tb, Hm/Sb
Genetic reagent (D. melanogaster)		This lab	N/A	w[1]; TM6, Tb, Hm/Sb
Genetic reagent (D. melanogaster)	Fub-1^attP	This paper	N/A	Available from Paul Schedl laboratory in Princeton university. Contact pschedl@princeton.edu
Genetic reagent (D. melanogaster)	Fab-7^attP50	This lab	N/A	Available from Paul Schedl laboratory in Princeton university. Contact pschedl@princeton.edu
Genetic reagent (D. melanogaster)	FLP	Bloomington Drosophila Stock Center	BDSC: 8727	P{w[+mC]=ovoFLP.R}M1A, w[*]
Genetic reagent (D. melanogaster)	Cre	Bloomington Drosophila Stock Center	BDSC: 851	y[1] w[67c23] P{y[+mDint2]=Crey}1b; D[*]/TM3, Sb[1]
Antibody	Anti- Abd-B (mouse monoclonal)	Developmental Studies Hybridoma Bank	1A2E9	IF(1:50)
Antibody	Anti-Ubx (mouse monoclonal)	Developmental Studies Hybridoma	FP3.38	IF(1:50)
Antibody	Anti-DsRed antibody (mouse monoclonal)	Santa Cruz Biotechnology	sc-390909	IF(1:50)
Antibody	Anti-GFP antibody (rabbit polyclonal)	Thermo Fisher Scientific	A-11122	IF(1:500)
Antibody	Anti-mouse Alexa Fluor 546 (goat polyclonal)	Thermo Fisher Scientific	A-11030	IF(1:500)
Antibody	Anti-rabbit Alexa Fluor 488 (goat polyclonal)	Thermo Fisher Scientific	A-11008	IF(1:500)
Chemical compound, drug	Paraformaldehyde 20% solution, EM Grade	Electron Microscopy Sciences	15713S
Chemical compound, drug	Formaldehyde, 16%, methanol free, Ultra Pure	Polysciences Inc	18814-10
Chemical compound, drug	Phosphate-buffered saline (10×) pH 7.4, RNase-free	Thermo Fisher	AM9624
Chemical compound, drug	Tween 20	Sigma	P1379
Chemical compound, drug	Triton X-100	Bio-Rad	161-0407
Chemical compound, drug	Tris base	Sigma	11814273001
Chemical compound, drug	Methanol	Fisher Chemical	203403
Chemical compound, drug	SSC, 20×	Thermo Fisher Scientific	15557044
Chemical compound, drug	Formamide	Thermo Fisher Scientific	17899
Chemical compound, drug	Dextran sulfate	Sigma	D8906
Chemical compound, drug	Salmon Sperm DNA	Thermo Fisher Scientific	AM9680
Chemical compound, drug	Ribonucleoside Vanadyl Complex	NEB	S1402S
Chemical compound, drug	Nuclease-free BSA	Sigma	126609
Chemical compound, drug	Triethylammonium acetate	Sigma	625718
Chemical compound, drug	dGTP (100 MM)	VWR	76510-208
Chemical compound, drug	dTTP (100 MM)	VWR	76510-224
Chemical compound, drug	Lonza NuSieve 3:1 Agarose	Thermo Fisher Scientific	BMA50090
Chemical compound, drug	T4 DNA ligase	NEB	M0202L
Chemical compound, drug	Biotin-11-dCTP	Jen Bioscience	NU-809-BIOX
Chemical compound, drug	Biotin-14-dATP	Jen Bioscience	NU-835-BIO14
Chemical compound, drug	Qubit dsDNA HS Assay Kit	Life Technologies Corporation	Q32851
Chemical compound, drug	Phase Lock Gel, QuantaBio - 2302830, Phase Lock Gel Heavy	VMR	10847-802
Chemical compound, drug	NEBNext Ultra II DNA Library Prep Kit for Illumina	NEB	E7645S
Chemical compound, drug	Ampure Xp 5 ml Kit	Thermo Fisher Scientific	NC9959336
Chemical compound, drug	Hifi Hotstart Ready Mix	Thermo Fisher Scientific	501965217
Chemical compound, drug	Dynabeads MyOne Streptavidin C1	Life Technologies Corporation	65001
Chemical compound, drug	cOmplete, EDTA-free Protease Inhibitor Cocktail	Sigma	11873580001
Chemical compound, drug	N,N-Dimethylformamide	Sigma	227056
Chemical compound, drug	Potassium acetate solution	Sigma	95843
Chemical compound, drug	DSG (disuccinimidyl glutarate)	Thermo Fisher Scientific	PI20593
Chemical compound, drug	T4 Polynucleotide Kinase – 500 units	NEB	M0201S
Chemical compound, drug	DNA Polymerase I, Large (Klenow) Fragment – 1000 units	NEB	M0210L
Chemical compound, drug	End-it DNA End Repair Kit	Thermo Fisher Scientific	NC0105678
Chemical compound, drug	Proteinase K recomb. 100 mg	Sigma	3115879001
Chemical compound, drug	Nuclease Micrococcal (s7)	Thermo Fisher Scientific	NC9391488
Chemical compound, drug	EGS (ethylene glycol bis(succinimidyl succinate))	Thermo Fisher Scientific	PI21565
Sequence-based reagent	Fub-1 sense probe set	Biosearch Technologies	N/A
Sequence-based reagent	Fub-1 antisense probe set	Biosearch Technologies	N/A
Software, algorithm	Fiji (ImageJ)	Schindelin et al., 2012		fiji.sc
Software, algorithm	NIS element	Nikon		https://www.microscope.healthcare.nikon.com/products/software/nis-elements/
Software, algorithm	GraphPad Prism 8	GraphPad Software		https://www.graphpad.com
Software, algorithm	HiGlass	Kerpedjiev et al., 2018		http://higlass.io/
Software, algorithm	bwa	Li and Durbin, 2009		https://bio-bwa.sourceforge.net/
Software, algorithm	samtools	Github/open source		https://samtools.github.io
Software, algorithm	pairsamtools	Github/open source; Goloborodko et al., 2023		https://github.com/open2c/pairtools
Software, algorithm	pairix	Github/open source; Lee et al., 2021		https://github.com/4dn-dcic/pairix
Software, algorithm	cooler	Abdennur et al., 2023		https://github.com/open2c/cooler
Software, algorithm	Miniconda	Anaconda		https://docs.conda.io/en/latest/miniconda.html
Software, algorithm	Snakemake	Github/open source		https://snakemake.github.io

Additional files

Supplementary file 1 Sequences of DNA fragments used. (a) The sequence of chimeric DNA fragments used for Fab-7 replacement experiments. (b) Sequences of Fub-1 sense smFISH probes covering 1983 bp region in bxd domain. (c) Sequences of Fub-1 antisense smFISH probes covering 1463 bp region in bxd domain.: https://cdn.elifesciences.org/articles/84711/elife-84711-supp1-v2.docx
Download elife-84711-supp1-v2.docx
MDAR checklist: https://cdn.elifesciences.org/articles/84711/elife-84711-mdarchecklist1-v2.pdf
Download elife-84711-mdarchecklist1-v2.pdf