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Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes

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Abstract

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the “open,” euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUURGAL4DBD and HP1GAL4DBD results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUURGAL4DBD had no measurable influence on mini-white expression, HP1GAL4DBD targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on the protein composition of target regions. HP1GAL4DBD but not SUURGAL4DBD could displace an open chromatin marker, CHRIZ, from the tethering sites.

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Acknowledgments

The authors are grateful to T. Kolesnikova and T. Dubatolova for their assistance with the experiments; C. Seum, P. Spierer, and Bloominghton Stock Center are gratefully acknowledged for providing valuable fly stocks; and the authors also thank S. Elgin for sharing the antibodies. This work was supported by the grant of the Russian Science Foundation (14-14-00934).

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, 630090, Russia

    Galina V. Pokholkova, Dmitry E. Koryakov, Alexey V. Pindyurin, Elena N. Kozhevnikova, Stepan N. Belyakin, Oleg V. Andreyenkov, Elena S. Belyaeva & Igor F. Zhimulev

  2. Novosibirsk State University, Novosibirsk, 630090, Russia

    Dmitry E. Koryakov, Alexey V. Pindyurin & Igor F. Zhimulev

  3. Institute of Cytology and Genetics SB RAS, Novosibirsk, 630090, Russia

    Alexey V. Pindyurin & Elena N. Kozhevnikova

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  1. Galina V. Pokholkova
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  2. Dmitry E. Koryakov
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  3. Alexey V. Pindyurin
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  4. Elena N. Kozhevnikova
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  7. Elena S. Belyaeva
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  8. Igor F. Zhimulev
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Correspondence to Elena S. Belyaeva.

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Suppl. Fig. 1

UAS-bearing constructs used in this work (adapted from Bellen et al. 2011) and their integration sites. Genomic coordinates according to the R5.56, FB2014_02 release (flybase.org) are shown (GIF 23 kb)

High Resolution Image (EPS 277 kb)

Suppl. Fig. 2

FISH mapping of UAS-3B, -8D, and -18B transgenes in polytene chromosomes. Mini-white DNA was used as a probe. Integration sites are shown by the arrows. In the case of UAS-3B, a second signal, distinct from 3B, is present at 3C (white arrow), which is due to hybridization of the probe to the endogenous white gene found at this location. Bar 5 μm (GIF 175 kb)

High Resolution Image (TIFF 9960 kb)

Suppl. Fig. 3

Immunolocalization of SUUR and HP1 in wild-type polytene chromosomes. SUUR is present in the pericentric heterochromatin (PH), nucleolus (N), and numerous intercalary heterochromatin sites. HP1 is largely concentrated in the pericentric heterochromatin and on the fourth chromosome and is less prominent in the region 31 and in telomeres (telomeres of the chromosomes 2R and X are indicated). Bar 5 μm (GIF 214 kb)

High Resolution Image (TIFF 13739 kb)

Suppl. Fig. 4

Immunostaining of polytene chromosomes from GAL4DBD > UAS-8D larvae with Myc-specific antibodies recognizing GAL4DBD protein. Tethering site 8D (arrow) is robustly labeled consistent with the recruitment of GAL4DBD to this genomic region (a). Anti-PCNA staining at different S phase stages (b–d) shows that binding of GAL4DBD to the UAS-8D site does not delay its replication timing. Much as in the wild-type chromosomes, this region is replicating early, and no replication at this site is observed during the mid- or late S phase. Bar 5 μm (GIF 138 kb)

High Resolution Image (TIFF 13570 kb)

Suppl. Fig. 5

Replication patterns around the tethering site at 3B3-4 (arrow) in the control chromosomes from UAS-3D larvae (a–c) and upon targeted recruitment of SUURGAL4DBD (d–f). Co-staining with anti-PCNA and anti-SUUR antibodies was used to determine the specific stage of S phase and visualize SUURGAL4DBD in the region of interest. Bar 5 μm (GIF 273 kb)

High Resolution Image (TIFF 17067 kb)

Suppl. Fig. 6

Replication patterns around the tethering site at 18B3 (arrow) in the control chromosomes from UAS-18D larvae (a–c) and upon tethering of SUURGAL4DBD (d–f). Co-staining with anti-PCNA and anti-SUUR antibodies was used to determine the specific stage of S phase and visualize SUURGAL4DBD in the region of interest. Bar 5 μm (GIF 242 kb)

High Resolution Image (TIFF 17081 kb)

Suppl. Fig. 7

BrdU incorporation pattern around the tethering site at 8D8-10 (arrow), as found at the late S phase in UAS-8D chromosomes in the absence (a) or presence (b) of tethered HP1GAL4DBD. Chromosomes were co-immunostained with antibodies against BrdU and HP1. In the absence of HP1GAL4DBD, the site of tethering completes replication by the end of the S phase, as opposed to the situation when HP1GAL4DBD is present, and replication at UAS-8D is still detectable at this late stage. Bar 5 μm (GIF 109 kb)

High Resolution Image (TIFF 7533 kb)

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Pokholkova, G.V., Koryakov, D.E., Pindyurin, A.V. et al. Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes. Chromosoma 124, 209–220 (2015). https://doi.org/10.1007/s00412-014-0491-8

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