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. 2024 Jul 8;52(12):6886-6905.
doi: 10.1093/nar/gkae393.

New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity

Vladimir Sokolov  1 , Olga Kyrchanova  1   2 , Natalia Klimenko  2 , Anna Fedotova  1 , Airat Ibragimov  2 , Oksana Maksimenko  2 , Pavel Georgiev  1
Affiliations

New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity

Vladimir Sokolov et al. Nucleic Acids Res. .

Abstract

In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Mzfp1 is a new C2H2 protein that interacts with CP190. (A) Phylogenetic tree with species of the Schizophora section of Diptera that contain Mzfp1 homologous proteins. A schematic representing the general organization of the Mzfp1 protein of Drosophila melanogaster is shown at the top. Two MADF domains (pink and blue pentagons) and C2H2 domains (orange rectangles) are indicated. The right panel indicates MADF domains that are contained in annotated proteins with the associated isoelectric points. The colors of the pentagons identify the MADF domains by their charges: blue = positively charged, pink = negatively charged. (B) Mapping of Mzfp1 domains interacting with CP190 in a yeast two-hybrid assay. Different fragments of Mzfp1 were fused to the GAL4 activating domain and tested for interaction with CP190 fused to the GAL4 DNA-binding domain. All Mzfp1 fragments were tested for the absence of interaction with the GAL4 DNA-binding domain alone (BD). The results are summarized in the columns on the right, with the ‘+’ and ‘–‘ signs referring to the presence and absence of interaction, respectively. Potential regions in Mzfp1 that interact with CP190 are indicated by red lines. (C) Total extracts from Drosophila S2 cells co-transfected with CP190 and Mzfp1-3xHA expressing plasmids were immunoprecipitated with antibodies against CP190 or nonspecific IgG as a negative control, and the immunoprecipitates were analyzed by Western blotting for the presence of HA-tagged proteins. CP190 and IgG immunoprecipitates are shown concentrated relative to the input by a factor of 20. Created with BioRender.com.
Figure 2.
Figure 2.
Mzfp1 binds to promoters of housekeeping genes. (A) Venn diagram for Mzfp1 binding sites (with and without the motif) obtained with antibodies against Mzfp1 obtained in rats and rabbits. (B) The consensus binding site (Mzfp1 motif) identified by ChIPMunk for the top 100 peaks from the combined set of Mzfp1 binding sites obtained using rat and rabbit antibodies. Below is the electrophoretic mobility shift assay for the recombinant full-sized Mzfp1 as well as for the cluster of C2H2 domains from Mzfp1 only. MBP-fused Mzfp1 was incubated with fluorescently labeled DNA fragments: four Mzfp1 binding sites (Mzfp1x4) labeled with Cy5 and four Zw5 binding sites (Zw5x4) labeled with fluorescein amidite (FAM) (used as a negative control). Signals were detected for FAM-labeled fragments at an excitation wavelength of 500 nm and an emission wavelength of 535 nm and for the Cy5-labeled fragment at an excitation wavelength of 630 nm and an emission wavelength of 700 nm. (C) The distribution of Mzfp1 binding sites (with and without a motif) by genomic elements in euchromatic and heterochromatic regions of the genome is shown on the left. On the right, the distribution of Mzfp1 sites between promoters of housekeeping genes and other Drosophila promoters is shown. (D) Colocalization of Mzfp1 sites with the binding sites for the M1BP, dCTCF, Su(Hw), Pita, ZIPIC, BEAF-32 and CP190 proteins. Only binding regions with motifs for all proteins except CP190 were considered in this analysis. The bars and numbers on the left correspond to all binding sites included in the analysis for the each protein.
Figure 3.
Figure 3.
The role of Mzfp1 in vivo. (A) A schematic representation of the CG1603f04743 mutation and the CG1603attP deletion. (B) Expression comparison using RNA-seq analysis in wild-type and CG1603attP/CG1603attPlarvae of euchromatic and heterochromatic groups of genes containing the Mzfp1 binding site with (‘with motif’) or without (‘NO motif’) the motif in the promoter region. The y1w1118; CG1603attP/CG1603attP larvae were collected from the cross between y1w1118; CG1603attP/СyO GFP males and females. The proportions of differentially expressed genes among those containing Mzfp1 binding sites in the promotor region (on the left) and the mean log-fold change for down-regulated, up-regulated and all differentially expressed genes containing Mzfp1 binding sites in the promotor region are shown on the right. (C) Schematic representation of the deletions in the Mzfp1 protein. A schematic showing the construct used to express wild-type Mzfp1 under the control of the Ubi-p63E (U) promoter in a transgenic Drosophila line. Green boxes – promoter and 5’UTR of Ubi-p63E gene; red box – 3’UTR with polyadenylation signal from dCTCF gene; yellow box – polyadenylation signal from virus SV40; violet boxes – 3xHA epitope. The black lines correspond to introns. The transgenes expressing different variants of Mzfp1 were tested in heterozygous state in combination with the CG1603f04743 homozygote. (D) Immunoblot analysis (8% SDS-PAGE) of protein extracts from transgenic flies expressing wild-type and deletion variants of Mzfp1 in adult flies (wt (Mzfp1wt-HA), Δ42 (Mzfp1Δ42-HA), ΔZF (Mzfp1 ΔZF-HA), ΔM1 (Mzfp ΔM1-HA), ΔM2 (Mzfp ΔM2-HA)) with antibodies against HA-epitope. Antibodies for lamin Dm0 were used as an internal control.
Figure 4.
Figure 4.
Distribution of the Mzfp1-HA variants (wt (Mzfp1wt-HA), Δ42 (Mzfp1Δ42-HA), ΔM1 (Mzfp ΔM1-HA), ΔM2 (Mzfp ΔM2-HA), and ΔZF (Mzfp1 ΔZF-HA)) on polytene chromosomes from larval salivary glands. The panels show the merged results of immunostaining of endogenous Mzfp1 and Mzfp1-HA variants (green, rabbit anti-Mzfp1 antibody; red, mouse anti-HA antibody). DNA was stained with DAPI (blue).
Figure 5.
Figure 5.
Binding specificity of Mzfp1 variants in adult flies. (A) Comparison of the Mzfp1 ChIP-Seq signal (log2(RPKM)) in adult Mzfp1wt-HA flies with anti-Mzfp1 and anti-HA antibodies at the combined set of binding sites identified by these antibodies. (B) Colocolization of Mzfp1 in Mzfp1wt-HA (wt), Mzfp1ΔM2-HA (ΔM2), Mzfp1Δ42-HA (Δ42), and Mzfp1ΔZF-HA (ΔZF) lines (on the top) and average ChIP-Seq signals (on the bottom) for wt, ΔM2, Δ42, ΔZF, and the pre-immune control (IgG) in the combined set of wt, ΔM2, Δ42 and ΔZF binding sites with and without the motif located in euchromatic and heterochromatic regions with anti-HA antibodies. (C) Colocolization of Mzfp1 in wt, ΔM2, Δ42 lines (on the top) and average ChIP-Seq signals (on the bottom) for Mzfp1 in wt, ΔM2 and Δ42 lines, and IgG in the combined set of wt, ΔM2 and Δ42 binding sites with and without the motif located in euchromatic and heterochromatic regions with anti-Mzfp1 antibodies. (D) Average ChIP-Seq signals for Mzfp1 in wt, ΔM2, Δ42 and ΔZF lines, and IgG in binding sites enriched in wt, ΔM2, Δ42 and ΔZF lines obtained with anti-HA antibodies. For the analysis with anti-HA antibodies only binding sites intersecting with ones identified in Mzfp1wt-HA (anti-HA) lines, y1w118 adults or embryos were considered in this analysis. (E) Average ChIP-Seq signals for Mzfp1 in wt, ΔM2 and Δ42 lines, and IgG in binding sites enriched in wt, ΔM2 and Δ42 lines obtained using anti-Mzfp1 antibodies.
Figure 6.
Figure 6.
Analysis of the functional roles of Mzfp1, Su(Hw), and Pita in the activity of F2411 inserted instead of the full Fub boundary. (A) Schematic representation of the BX-C including Ubx, abd-A, and Abd-B genes and the Fub boundary. The Ubx, abd-A and Abd-B genes are shown in orange, blue, and green horizontal arrows, respectively. Their regulatory regions are marked with the same color-coded bars. The boundaries (Fab-1, Fub (Fab-2), Fab-3, Fab-4, Mcp, Fab-6, Fab-7 and Fab-8) that separate segment-specific regulatory domains (abx/bx, bxd/pbx – regulate Ubx, iab-2, iab-3 and iab-4 – abd-A and iab-5, iab-6, iab-7 and iab-8,9 – Abd-B) are indicated by vertical black bars. A schematic of the F2attP deletion is shown below. The Fub boundary has two hypersensitive regions (gray boxes). F2411 is shown as an orange bar with marked Mzfp1 (magenta oval), Pita (blue oval), and Su(Hw) (green oval) binding sites. F2177 is shown as an yellow bar with marked Su(Hw) (green oval) and dCTCF (red oval) binding sites. Mutated sites are shown as an erased region in place of the corresponding binding sites. The endpoints of the F2attP deletion are indicated by breaks in the black lines. The F2411fragments used in the experiments and the results of deletion and rescue are shown at the bottom: (+) – the fragment has insulator activity in embryos/adults; A1(PS6) and A2(PS7) segments have the wt phenotype; (-) the fragment has no insulator activity; A1(PS6) transforms into A2(PS7); (±) – the fragment has partial insulator activity in some cells; A1(PS6) has several characteristics of the A2(PS7). (B) Morphology of the A1 and A2 abdominal segments (numbered) under different F2attP replacement schemes. In wt the A1 sternite is absent; the tergite has a specific form and is thinned towards the center, with no long bristles. A2 comprises a quadrilateral sternite with vertically oriented bristles and a tergite with a pigmented stripe and long bristles. In the F2attP deletion, the A1 tergite is wider; its posterior margin is pigmented and covered in large bristles as in the A2; the A1 sternite is covered in bristles and resembles the sternite in A2. The signs of the A1→A2 transformation are indicated by red arrowheads. (C) Expression of the abd-A gene in embryos with different F2attP replacements. Each panel shows a confocal image of the embryo at stage 14 stained with antibodies against the Abd-A protein (magenta). The parasegments are numbered from 5 to 14 on the right side of each embryo image. The number of the approximately corresponding adult segment is indicated on the left. In wt embryos, abd-A is inactive in PS5 and PS6, whereas it is active in PS7-PS12. In F2attP embryos, Abd-A was detected in PS6. The yellow arrowheads indicate the ectopic expression of abd-A.
Figure 7.
Figure 7.
Binding of Mzfp1, Su(Hw), and Pita to F2411 and mutant derivatives in two-day-old adult males. (A) Tracks for Mzfp1, dCTCF, Pita, Su(Hw), and CP190 binding profiles at the Fubboundary via a ChIP-Seq analysis using embryos. (B) Binding of Mzfp1 (magenta), Pita (dark blue), Su(Hw) (green), and CP190 (yellow) with F2411, F2ΔM, F2ΔP, F2ΔS, F2ΔMΔP, F2ΔMΔS and F2ΔPΔS elements. The results of ChIPs are presented as the percentage of input DNA, normalized against a positive genomic site: 62D, for Su(Hw), 50E, for Pita and 94C, for Mzfp1 and CP190 binding. The error bars indicate SDs of quadruplicate PCR measurements from three independent biological samples of chromatin.
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References

    1. Haberle V., Stark A.. Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 2018; 19:621–637. - PMC - PubMed
    1. Sikorska N., Sexton T.. Defining functionally relevant spatial chromatin domains: it is a TAD complicated. J. Mol. Biol. 2020; 432:653–664. - PubMed
    1. Sloutskin A., Shir-Shapira H., Freiman R.N., Juven-Gershon T.. The core promoter is a regulatory hub for developmental gene expression. Front. Cell Dev. Biol. 2021; 9:666508. - PMC - PubMed
    1. Levo M., Raimundo J., Bing X.Y., Sisco Z., Batut P.J., Ryabichko S., Gregor T., Levine M.S.. Transcriptional coupling of distant regulatory genes in living embryos. Nature. 2022; 605:754–760. - PMC - PubMed
    1. Nollmann M., Bennabi I., Götz M., Gregor T.. The impact of space and time on the functional output of the genome. Cold Spring Harb. Perspect. Biol. 2021; 14:a040378. - PMC - PubMed

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