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. 2020 May 7;48(8):4214-4229.
doi: 10.1093/nar/gkaa158.

A novel transcriptional cascade is involved in Fzr-mediated endoreplication

Wenliang Qian  1   2 , Zheng Li  1   2 , Wei Song  3   4 , Tujing Zhao  1   2 , Weina Wang  1   2 , Jian Peng  1   2 , Ling Wei  5 , Qingyou Xia  1   2 , Daojun Cheng  1   2
Affiliations

A novel transcriptional cascade is involved in Fzr-mediated endoreplication

Wenliang Qian et al. Nucleic Acids Res. .

Abstract

Endoreplication, known as endocycle, is a variant of the cell cycle that differs from mitosis and occurs in specific tissues of different organisms. Endoreplicating cells generally undergo multiple rounds of genome replication without chromosome segregation. Previous studies demonstrated that Drosophila fizzy-related protein (Fzr) and its mammalian homolog Cdh1 function as key regulators of endoreplication entrance by activating the anaphase-promoting complex/cyclosome to initiate the ubiquitination and subsequent degradation of cell cycle factors such as Cyclin B (CycB). However, the molecular mechanism underlying Fzr-mediated endoreplication is not completely understood. In this study, we demonstrated that the transcription factor Myc acts downstream of Fzr during endoreplication in Drosophila salivary gland. Mechanistically, Fzr interacts with chromatin-associated histone H2B to enhance H2B ubiquitination in the Myc promoter and promotes Myc transcription. In addition to negatively regulating CycB transcription, the Fzr-ubiquitinated H2B (H2Bub)-Myc signaling cascade also positively regulates the transcription of the MCM6 gene that is involved in DNA replication by directly binding to specific motifs within their promoters. We further found that the Fzr-H2Bub-Myc signaling cascade regulating endoreplication progression is conserved between insects and mammalian cells. Altogether, our work uncovers a novel transcriptional cascade that is involved in Fzr-mediated endoreplication.

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Figures

Figure 1.
Figure 1.
Specific knockdown of Fzr expression in Drosophila salivary gland blocks endoreplication and results in the initiation of CycB transcription. (A) RNAi-mediated Fzr knockdown driven by salivary gland-specific Sg-Gal4 resulted in a significant reduction in the gland size. At 120 h AEL, salivary glands from control and Fzr knockdown Drosophila were dissected and stained with DAPI. Two Fzr RNAi lines targeting different sequences were used in this experiment. Fzr-i#1: UAS-Fzr RNAi line from VDRC (#V25550); Fzr-i#2: UAS-Fzr RNAi line from TsingHua Fly Center (#TH2015000745.S). Scale bar, 180 μm. (B) C-value was quantified by DAPI fluorescence. The salivary glands from wandering Drosophila larvae at 120 h AEL were fixed and stained with DAPI. The integrated DAPI intensity was used to measure the DNA content. (C) Quantification of the DNA content in the salivary glands of Drosophila larvae at 120 h AEL. DNA was extracted from the indicated salivary gland cells and quantified via absorbance analysis. (D, E) EdU staining of DNA replication. In the salivary glands of larvae at 96 h AEL, the nuclei of most endocycling cells in the salivary glands as control could be strongly stained with EdU, indicating that DNA synthesis is ongoing. However, no replication signals were detected in salivary gland cells with Fzr knockdown. Scale bar, 30 μm. (F–J) Fzr knockdown in the salivary glands causes an accumulation of the CycB protein (F, G) and an appearance of the CycB mRNA (H–J) at 96 h AEL. The mRNA level was measured by fluorescence in situ hybridization (H, I) and RT-qPCR(J). Scale bar, 30 μm. (K)Fzr overexpression in Drosophila S2 cells significantly downregulated the transcription of the CycB gene. Data are presented as mean ± SE (error bars). For the significance test: ***P < 0.001 versus control. OE, overexpression. AEL, after egg laying.
Figure 2.
Figure 2.
Fzr expression changes affect the transcription of transcription factor gene Myc and Myc is required for inhibition of CycB transcription in Drosophila salivary gland. (A) Transcriptome analysis of expression changes of several transcription factor genes following Fzr overexpression in Drosophila S2 cells. Normalized FPKM value was used for measuring relative expression level. Myc was upregulated following Fzr overexpression. Data are presented as mean + SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (B) Fzr overexpression in S2 cells promoted mRNA transcription and protein expression of the Myc gene. (C) Fzr knockdown in the salivary glands reduced mRNA transcription and protein expression of the Myc gene at 96 h AEL. (D–H) Myc knockdown in the salivary glands causes an accumulation of the CycB mRNA (D, E and H) and the CycB protein (F, G) at 96 h AEL. (I) Myc overexpression in Drosophila S2 cells significantly downregulated the transcription of the CycB gene. Data are presented as mean ± SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 versus control. OE, overexpression. AEL, after egg laying. Scale bar, 30 μm.
Figure 3.
Figure 3.
Myc functions as a downstream effector of Fzr modulation during endoreplication in Drosophila salivary gland. (A–H) Epistasis analysis revealed that Myc overexpression in the salivary glands moderately rescued the effects of Fzr knockdown on gland size (A–C), C-value (D), and DNA content (E) at 120 h AEL as well as DNA replication (F–H) at 96 h AEL. (I–K) Fzr knockdown-induced CycB expression in the salivary glands was abrogated by salivary gland-specific Myc overexpression at 96 h AEL. Data are presented as mean ± SE (error bars). For the significance test: **P < 0.01, ***P < 0.001 versus control. OE, overexpression. AEL, after egg laying. Scale bar, 30 μm.
Figure 4.
Figure 4.
Fzr enhances H2B ubiquitination within the promoter of the Myc gene. (A) Co-IP analysis revealed that Fzr interacts with H2B. (B) Following nucleus isolation, micrococcal nuclease digestion, and nucleoprotein extraction, Co-IP experiments with nucleoproteins were performed and then confirmed the interaction between Fzr and chromatin-associated H2B. (C) GST-pull down assay showed a direct interaction between Fzr and H2B. (D, E) Fzr promotes H2B ubiquitination. The level of ubiquitinated H2B (H2Bub) was increased following Fzr overexpression in Drosophila S2 cells (D) and was decreased after Fzr knockdown in the salivary glands at 96 h AEL (E). (F) An in vivo ubiquitination assay in S2 cells confirmed that Fzr promotes H2B mono-ubiquitination. Ub-K0 is a mutated ubiquitin that all lysines are mutated to arginines and only mediates mono-ubiquitination. The nucleoproteins were extracted from S2 cells co-overexpressing several designed molecules and were then used for Co-IP analysis with anti-Myc tag antibody. (G, H) ChIP-qPCR assays in Fzr-overexpressing S2 cells by using anti-H2Bub antibody (G) and anti-Fzr antibody (H) revealed that the amounts of ubiquitinated H2B and Fzr that binds to the promoter region of the Myc gene were elevated after Fzr overexpression. Data are presented as mean ± SE (error bars). For the significance test: *P < 0.05, **P < 0.01 versus control. OE, overexpression. AEL, after egg laying.
Figure 5.
Figure 5.
Myc promotes the transcription of the MCM6 gene involving in DNA replication. (A, B) Effects of Myc expression change on the transcription of all members of the MCM complex that function as components of preRCs and to initiate DNA replication. RT-qPCR analysis showed that the transcription of the MCM6 gene was decreased after Myc knockdown (A) but was increased following Myc overexpression (B) in the salivary glands at 96 h AEL. (C–F) Immunostaining analysis confirmed that Myc promoted the expression of the MCM6 protein. The MCM6 protein level in the salivary glands was downregulated after Myc knockdown at 96 h AEL (C, D) but was upregulated following Myc overexpression at 120 h AEL (E, F). (G–I) Fzr knockdown reduced MCM6 expression in the salivary glands and this reduction was diminished by simultaneous Myc overexpression at 96 h AEL. (J-L)Myc overexpression-induced enhancement in DNA replication was impaired by MCM6 knockdown in salivary glands at 120 h AEL. Data are presented as mean ± SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 versus control. OE, overexpression. AEL, after egg laying. Scale bar, 30 μm.
Figure 6.
Figure 6.
Myc regulates the transcription of the CycB and MCM6 genes by directly binding to specific motif within their promoters. (A) Schematic diagram of the Myc binding peaks and potential E-box motifs for Myc binding within the promoters of the CycB and MCM6 genes. One binding peak is located in the region from -921 to -461 within the promoter upstream of the translational start site of the CycB gene and one potential E-box for Myc binding is located within this region from -838 to -833. One binding peak exists within the region from –359 to +221 around the translational start site of the MCM6 gene and there is one potential E-box within this region from –83 to –78. (B–C′) ChIP-PCR and ChIP-qPCR assays verified the direct binding of Myc to the promoters of the CycB and MCM6 genes in Drosophila salivary glands (B, B’) and S2 cells with Myc overexpression (C, C’). (D, D′) Electrophoretic mobility shift assay (EMSA) confirmed that Myc can directly bind to specific E-box motifs for Myc binding within the promoters of the CycB (D) and MCM6 genes (D’). Anti-Myc antibody competitively impaired the binding of Myc to the probes targeting E-box motifs. (E, E′) Luciferase reporter analyses revealed that Myc inhibited and promoted activities of the CycB promoter (E) and the MCM6 promoter (E’), respectively. P1, complete promoters of the CycB and MCM6 genes containing potential E-box. P2, truncated promoters of the CycB and MCM6 genes without E-box. Data are presented as mean ± SE (error bars). For the significance test: **P < 0.01 versus control. OE, overexpression.
Figure 7.
Figure 7.
The Fzr-H2Bub-Myc signaling cascade is conserved between insect and mammalian cells. (A–C) Effects of the overexpression of the human Fzr (HsFzr) gene in human HEK293-FT cells. HsFzr overexpression not only increased the size of the cell and nucleus (A-B’), but also inhibited HsCycB expression and promoted both HsMyc expression and H2B ubiquitination (C), which is similar to the effects of the overexpression of the Drosophila Fzr (DmFzr) gene in Drosophila S2 cells. (D) Overexpression of the human Myc (HsMyc) gene in HEK293-FT cells inhibited HsCycB expression. (E–F’) Ectopic overexpression of the HsFzr gene in Drosophila S2 cells also increased the size of the cell and nucleus. (G) Ectopic overexpression of human HsFzr in Drosophila S2 cells inhibited DmCycB expression, promoted DmMCM6 expression, and enhanced both DmMyc expression and H2B ubiquitination. (H) Ectopic overexpression of human HsMyc in S2 cells inhibited DmCycB expression and promoted DmMCM6 expression. OE, overexpression. Hs, Homo sapiens; Dm, Drosophila melanogaster. Scale bar, 5 μm.
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