Drosophila SUMM4 complex couples insulator function and DNA replication timing control

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    This paper will be of interest to those studying DNA replication in the context of chromatin and development. This important study uncovers a new interaction partner for the chromatin protein SuUR and tries to understand how this complex (SUMM4) functions to control under-replication in polytene chromosomes. While the experiments are of high quality and carefully controlled, the data currently do not fully support all the conclusions, particularly as they relate to conclusions about DNA replication timing.

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Abstract

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under- replication of late-replicating intercalary heterochromatin in polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo . SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse under-replication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for replication timing that does not depend on an asynchronous firing of replication origins.

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  1. eLife assessment

    This paper will be of interest to those studying DNA replication in the context of chromatin and development. This important study uncovers a new interaction partner for the chromatin protein SuUR and tries to understand how this complex (SUMM4) functions to control under-replication in polytene chromosomes. While the experiments are of high quality and carefully controlled, the data currently do not fully support all the conclusions, particularly as they relate to conclusions about DNA replication timing.

  2. Reviewer #1 (Public Review):

    Andreyeva et al. developed a novel purification/mass spec approach to identify SuUR-associated proteins. From this biochemical tour de force, they identify a complex consisting of the insulator-associated protein Mod(Mdg4) and SuUR that they term, SUMM4. They show that this complex (at least SuUR) has ATPase activity, which is an exciting result was no known biochemical activity associated with SuUR. Given SuUR's function in the under-replication of Drosophila salivary glands, the authors show that SuUR and Mod(Mdg4) at least partially localize on polytene chromosomes and that SuUR displays at least a partial dependence on Mod(Mdg4) for localization to IH, but not PH regions. Finally, using two independent genetic reporters, they show that SuUR itself has an insulator function, which is a new function for SuUR and exciting as it is likely a diploid cell-specific function for SuUR. The authors then attempt to show the Mod(Mdg4) functions in under-replication. Unfortunately, under-replication is minimally, if at all, changed in the Mod(Mdg4) mutant. While the authors bring up several possible scenarios of why this could be, it is still uncertain whether Mod(Mdg4) has a direct effect on under-replication.

    Strengths:
    The authors developed a very useful strategy to identify protein interactions through multiple purification steps using mass spectrometry. This approach can be applied to different systems and will be generally useful to the community. Through this approach, they provide very compelling data that SuUR and Mod(Mdg4) form a complex. Furthermore, the experiments all have been rigorously performed and the data is of high quality.

    Weaknesses:
    The way the paper is written, its main focus is on under-replication. What the authors were not able to conclusively demonstrate is whether Mod(Mdg4) functions in under-replication.

  3. Reviewer #2 (Public Review):

    This paper from the Fyodorov lab reports the isolation of a native protein complex of SUUR, a Drosophila SNF2-related factor, in a complex with Mdg4, an established chromatin boundary protein. The discovery of this native complex, called SUMM4, was enabled by the development of a mass spec-linked proteomic analysis of fractions from an unbiased, conventional multi-step chromatographic purification of low-abundance protein complexes. The authors validate the native interactions by co-immunoprecipitation and show further with recombinant proteins that SUUR displays ATPase activity, a property not previously shown, and which is stimulated by Mdg4. From a functional perspective, authors demonstrate that both components SUUR and Mdg4 mediate activities of the Drosophila gypsy insulator that blocks enhancer-promoter interactions and acts as a heterochromatin-euchromatin barrier, and moreover, has a role in the under-replication of intercalary heterochromatin.

    Overall, this work is a substantial contribution to the field in two respects. First, it provides a new approach to the identification of novel native complexes that are of low abundance and difficult to isolate and identify by conventional biochemistry and mass spectrometry. Second, the interaction between Mdg4 and SUUR is novel and offers an ATP-driven pathway to be further investigated for understanding the mechanism of insulator (gypsy) function. Together, these advances are supported by the compelling quality and quantity of data. However, the paper does not read smoothly and can benefit from rewriting for readers who are not familiar with mass-spec proteomics or Drosophila biology.

  4. eLife assessment

    This paper will be of interest to those studying DNA replication in the context of chromatin and development. This important study uncovers a new interaction partner for the chromatin protein SuUR and tries to understand how this complex (SUMM4) functions to control under-replication in polytene chromosomes. While the experiments are of high quality and carefully controlled, the data currently do not fully support all the conclusions, particularly as they relate to conclusions about DNA replication timing.

    We appreciate a positive evaluation of our work. We agree that the relevance of under-replication phenomenon to the establishment of late replication in dividing cells has only been established based on circumstantial evidence. In the revised manuscript, we expand the explanation of this relationship and discuss limitations of the endoreplication model as applied to understanding of late DNA replication in the cell cycle of diploid cells. We also edited the abstract to soften our conclusions. We believe that the improvements made in the revised manuscript produced a more stringent alignment between our data and the conclusions.

    Reviewer #1 (Public Review):

    Andreyeva et al. developed a novel purification/mass spec approach to identify SuUR-associated proteins. From this biochemical tour de force, they identify a complex consisting of the insulator-associated protein Mod(Mdg4) and SuUR that they term, SUMM4. They show that this complex (at least SuUR) has ATPase activity, which is an exciting result was no known biochemical activity associated with SuUR. Given SuUR's function in the under-replication of Drosophila salivary glands, the authors show that SuUR and Mod(Mdg4) at least partially localize on polytene chromosomes and that SuUR displays at least a partial dependence on Mod(Mdg4) for localization to IH, but not PH regions. Finally, using two independent genetic reporters, they show that SuUR itself has an insulator function, which is a new function for SuUR and exciting as it is likely a diploid cell-specific function for SuUR. The authors then attempt to show the Mod(Mdg4) functions in under-replication. Unfortunately, under-replication is minimally, if at all, changed in the Mod(Mdg4) mutant. While the authors bring up several possible scenarios of why this could be, it is still uncertain whether Mod(Mdg4) has a direct effect on under-replication.

    Strengths:
    The authors developed a very useful strategy to identify protein interactions through multiple purification steps using mass spectrometry. This approach can be applied to different systems and will be generally useful to the community. Through this approach, they provide very compelling data that SuUR and Mod(Mdg4) form a complex. Furthermore, the experiments all have been rigorously performed and the data is of high quality.

    Weaknesses:
    The way the paper is written, its main focus is on under-replication. What the authors were not able to conclusively demonstrate is whether Mod(Mdg4) functions in under-replication.

    We thank the Reviewer for a positive evaluation of our work, specifically the biochemical and cytological results. Unfortunately, this Reviewer was less convinced by our conclusions about the role of Mod(Mdg4) in regulation of under-replication. However, we believe that our data strongly implicate Mod(Mdg4) in under-replication:

    1. Although SuUR is considered a bona fide suppressor of under-replication, its mutation does not fully restore DNA copy numbers in under-replicated regions of polytene chromosomes but, rather, by ~78% on average (Table 1). Although the mutation of mod(mdg4) produces a weaker recovery (~26% on average, Table 1), it is still robust and statistically significant. Presently, there is only one other mutant (Rif1) known to restore DNA copy numbers at most under-replicated regions in salivary gland polytene chromosomes.

    2. DNA copy numbers in SuUR and Rif1 mutants, which are homozygous viable and fertile, are measured in L3 larvae produced from crosses of homozygous parents, i.e. in the absence of maternally contributed gene products. In contrast, mod(mdg4) is essential for viability, and the DNA copy numbers have to be measured in homozygotes that have Mod(Mdg4) protein and RNA loaded by heterozygous mothers. Since endoreplication initiates before the maternal product is exhausted, it limits the observed suppression. However, when we directly compare zygotic functions of SuUR and mod(mdg4) by analyzing the progeny of heterozygous mod(mdg4)/+ and SuUR/+ parents, they appear indistinguishable.

    3. Finally, we demonstrate that Mod(Mdg4) is essential for the proper loading of SUUR in polytene chromosomes, thus implicating it as a direct, SUUR-dependent effector of late DNA replication.

    In the revised manuscript, we provide a clearer explanation of our results. We hope that our arguments and modifications of the manuscript will alleviate the Reviewer’s concerns.

    Reviewer #2 (Public Review):

    This paper from the Fyodorov lab reports the isolation of a native protein complex of SUUR, a Drosophila SNF2-related factor, in a complex with Mdg4, an established chromatin boundary protein. The discovery of this native complex, called SUMM4, was enabled by the development of a mass spec-linked proteomic analysis of fractions from an unbiased, conventional multi-step chromatographic purification of low-abundance protein complexes. The authors validate the native interactions by co-immunoprecipitation and show further with recombinant proteins that SUUR displays ATPase activity, a property not previously shown, and which is stimulated by Mdg4. From a functional perspective, authors demonstrate that both components SUUR and Mdg4 mediate activities of the Drosophila gypsy insulator that blocks enhancer-promoter interactions and acts as a heterochromatin-euchromatin barrier, and moreover, has a role in the under-replication of intercalary heterochromatin.

    Overall, this work is a substantial contribution to the field in two respects. First, it provides a new approach to the identification of novel native complexes that are of low abundance and difficult to isolate and identify by conventional biochemistry and mass spectrometry. Second, the interaction between Mdg4 and SUUR is novel and offers an ATP-driven pathway to be further investigated for understanding the mechanism of insulator (gypsy) function. Together, these advances are supported by the compelling quality and quantity of data. However, the paper does not read smoothly and can benefit from rewriting for readers who are not familiar with mass-spec proteomics or Drosophila biology.

    We thank the Reviewer for a positive evaluation of our work. To improve clarity, we made several modifications of our manuscript as requested by the Reviewer.