DNA–RNA interactions are critical for chromosome condensation in

Zhong Qiana,1, Victor B. Zhurkinb, and Sankar Adhyaa,1

aLaboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Sankar Adhya, September 25, 2017 (sent for review June 23, 2017; reviewed by Yan Jie and Jörg Vogel) Bacterial chromosome () conformation dictates faithful Results regulation of gene . The conformation is condition- Bulged Hairpin Structures Are Critical for the naRNA4 Function in DNA dependent and is guided by several nucleoid-associated pro- Condensation. Atomic force microscopy (AFM) was used to teins (NAPs) and at least one nucleoid-associated noncoding RNA, demonstrate HU and naRNA4-mediated DNA–DNA interac- naRNA4. Here we investigated the molecular mechanism of how tions, a mechanism of chromosome condensation first indicated naRNA4 and the major NAP, HU, acting together organize the by chromosome conformation capture assays (3C) to detect seg- – chromosome structure by establishing multiple DNA DNA contacts mental contacts in chromosomes (12, 15). Previously, we found (DNA condensation). We demonstrate that naRNA4 uniquely acts that naRNA4 may not require extensive homology to target DNA – by forming complexes that may not involve long stretches of DNA in the condensation process (12). Using AFM assays, we de- RNA hybrid. Also, uncommonly, HU, a chromosome-associated pro- termined the required feature(s) of naRNA4 in DNA interactions – tein that is essential in the DNA RNA interactions, is not present in in terms of primary and secondary structures. naRNA4 can form a the final complex. Thus, HU plays a catalytic (chaperone) role in the secondary structure containing two hairpin motifs (Y and Z2), naRNA4-mediated DNA condensation process. each of which contains a mismatched segment (bulge) (Fig. S1). We found that both Y and Z2 motifs are needed; deletion of ei- chromosome structure | DNA–RNA interaction | noncoding RNA | ther one made naRNA4 inactive for DNA condensation (12). Escherichia coli | HU protein Here we constructed several naRNA4 variants and used them in AFM assays to detect DNA–RNA interactions. The following hromosome organization is a focus of intense research in RNA variants were used: (i) RNA without bulges in both hair- Cboth prokaryotic and eukaryotic organisms. It is clear now pins (naRNA4flat); (ii) RNA with two Y hairpins (naRNA4YY); ZZ that chromosomal structures have significant influence on gene (iii) RNA with two Z2 hairpins (naRNA4 ); and (vi) RNA with – ZY transcription (1 3). In transcription regulation, the DNA does the Y and Z2 positions exchanged (naRNA4 ). The predicted sec- not just provide sequence information for binding and action of ondary structures are shown in Fig. S1. We found that, similar to wild- RNA polymerase and transcription factors; the conformation of YY ZZ

type naRNA4 that condenses DNA, the naRNA4 ,naRNA4 ,and GENETICS the DNA also plays an important role in faithfully regulating naRNA4ZY constructs were also able to condense DNA. However, transcription of specific genes when appropriate. It has been naRNA4flat without bulges could not make DNA condense under proposed that the Escherichia coli chromosome has a condition- similar conditions (Fig. 1 and Fig. S2 A–L). These results suggest that dependent defined structure (4). A 4.6-Mbp DNA is packaged the naRNA4 constructs must have bulged hairpins for the DNA inside a small cellular volume, and this packaging imparts a condensation function. defined structure to the DNA. Several factors contribute to the DNA packaging. First, the chromosomal DNA is mostly Significance supercoiled, which contributes to its condensation in terms of volume (5, 6). Second, several nucleoid-associated basic pro- This study focuses on the molecular mechanisms of a non- teins (NAPs), such as HU, HNS, CspE, CBP, FIS, etc., par- coding RNA-mediated chromosome organization in Escher- ticipate in chromosome condensation (7). Some of these ichia coli. We show for the first time that noncoding RNA, proteins, such as HU and HNS, modulate gene transcription naRNA4, a critical element at least in part of chromosome specifically as well as globally (1, 8–10). More recently, some structural organization (DNA condensation), may form DNA– noncoding RNAs have also been shown to be involved in RNA complexes in an uncommon way. Surprisingly, we found chromosome structure in E. coli (11, 12). We previously dem- that HU protein catalyzes the DNA–RNA interactions without onstrated that one of them, naRNA4, encoded by a DNA- “ ” REP being part of the final complexes. There are several im- repeat element ( 325), can connect two DNA elements portant examples of noncoding RNA playing critical roles in (dyad symmetry sequences) in the chromosome in the presence eukaryotic chromatin functioning, e.g., heterochromatin as- of HU protein to form DNA loops (12). sembly, gene silencing, and X-inactivation, although nothing Here, we investigated the molecular mechanisms of naRNA4- is known about the nature of DNA–RNA interaction(s) in these mediated chromosome condensation (the product sometimes is processes. Our findings will be of great interest in the chro- referred to as DNA condensate) and the role of HU in the matin field, providing mechanistic insights into the large-scale process. We show that the naRNA4, taking advantage of po- organization of DNA. tential secondary structure, can form DNA–RNA complexes provided that the DNA is supercoiled, converting palindromic Author contributions: Z.Q., V.B.Z., and S.A. designed research; Z.Q. performed research; sequences into cruciform structures leading to chromosome Z.Q., V.B.Z., and S.A. analyzed data; and Z.Q., V.B.Z., and S.A. wrote the paper. condensation in E. coli. We note that naRNA4 is different from Reviewers: Y.J., National University of Singapore; and J.V., University of Würzburg. the long noncoding RNAs (lncRNAs), which are involved in The authors declare no conflict of interest. chromosome structure in the eukaryotic cells (13, 14); lncRNAs Published under the PNAS license. interact with DNA-bound proteins but not directly with DNA. 1To whom correspondence may be addressed. Email: [email protected] or qianz@ Surprisingly, HU protein participating in this process is not a mail.nih.gov. “ ” – part of the final DNA RNA complex, suggesting a chaperon- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. like role of HU. 1073/pnas.1711285114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1711285114 PNAS | November 14, 2017 | vol. 114 | no. 46 | 12225–12230 Downloaded by guest on October 2, 2021 Note that bacterial DNA has a highly dynamic topology with signal, confirming the existence of DNA in the complexes that relaxed or supercoiled states (16, 17). We observed that negative bind to the antibody (Fig. 2, Top). The results validate the IPP supercoiling of DNA targets is critical for naRNA4-HU–mediated method. We also performed the IPP assays using the naRNA4 DNA condensate formation; no DNA interactions were observed variants (naRNA4flat, naRNA4YY, naRNA4ZZ,andnaRNA4ZY). when relaxed DNA, pSA508rel, was used in the assays (Fig. 1 and Consistent with AFM observations, PCR signals were observed in Fig. S2 N and O). the complexes with naRNA4YY, naRNA4ZZ, and naRNA4ZY but not with naRNA4flat (Fig. 2). We also tested wild-type naRNA4 DNA–naRNA4 Complex Formation Is Critical for DNA Condensation. with relaxed DNA (pSA508rel)aswellaswild-type Earlier, we proposed various putative models for naRNA4 and naRNA4 and supercoiled plasmid DNA followed by RNaseH HU-mediated DNA–DNA interactions both with and without treatment before IP. Clearly, PCR amplification failed in both formation of DNA–RNA complexes (12). To check if there was cases, suggesting that the complex may contain some kind of any DNA–RNA hybrid formation in the DNA condensate, we DNA–RNA hybrid (Fig. 2). treated the condensates with RNaseH, a sequence nonspecific We further confirmed the above findings about DNA conden- endonuclease that cleaves only RNA in a DNA–RNA hybrid, sation by IP in combination with fluorescence microscopy (IPFM, before AFM imaging. Interestingly, the RNaseH-treated samples flowchart shown in Fig. S3B). We used Cy-3–labeled naRNA4 and did not show any DNA condensation, indicating that the DNA aDNA–RNA hybrid-specific antibody (S9.6), which was coated condensate may contain some form of DNA–RNA hybrid (Fig. 1 with silica beads. In this assay, the beads capturing the potential – and Fig. S2M). DNA RNA complexes showed Cy-3 fluorescence under fluores- To corroborate this observation, we developed an immuno- cence microscopy (FM). Indeed, we observed fluorescent beads in the assays with naRNA4, naRNA4YY, naRNA4ZZ,and precipitation assay in combination with PCR [IP-PCR (IPP) ZY assay] using a specific antibody that could recognize DNA–RNA naRNA4 (Fig. 3). Both our current data and a previous report hybrids, S9.6 (18–20). The protocol is outlined in Fig. S3A. Any (21) confirmed that the silica beads used are free of any auto- DNA–naRNA4 complexes formed would bind to the antibody fluorescence. The frequency of the observed beads with fluores- conjugated to the beads. Using primers specific to the plasmid cent signal among all counted beads varies from 20 to 30% (Table 1). We also detected the Cy-3 signals in assays with naRNA4 and DNA, we could amplify DNA fragments in the eluates from the YY ZZ ZY beads. An amplification of the DNA signal would indicate the its variants (naRNA4 ,naRNA4 ,andnaRNA4 )inthe absence of HU protein at 2–5% levels, which are reproducibly existence of DNA–RNA complexes in the condensates. These lower than the frequency observed in the presence of HU (Fig. 3 results with the wild-type naRNA4 in the presence of plasmid and Table 1). This suggests that fluorescence assays used to detect pSA508SC DNA and HU clearly showed a PCR amplification the DNA–RNA complex is more sensitive than either AFM or IPP assay. As expected from AFM and IPP assays, we did not detect any fluorescent signals with naRNA4flat or with relaxed DNA (pSA508rel). Next, we treated naRNA4 mixed with pSA508SC in the presence of HU with RNaseH; we did not detect any Cy-3 signal in the microscope in this case (Fig. 3). All of the results presented here show that DNA–naRNA4 interactions are critical for condensation, and the presence of HU protein, although not indispensable, enhances the condensation process.

Cruciform Structure Is the Target in DNA for naRNA4 and HU Action. Dyad symmetry sequences in plasmid DNA form cruciform structures when DNA is supercoiled (22). Cruciform structures also show high affinity and specificity for the HU protein (23– 26). The idea that DNA cruciforms are the targets of naRNA4 and HU is supported by the fact that, as mentioned above, the supercoiled but not relaxed DNA showed DNA condensation. To test whether only the cruciform structures, or DNA superhelicity, or both are needed for condensation, we synthesized a cruciform DNA (syn-crDNA) with free 5′ and 3′ ends (no supercoiling) (Fig. 4A) and used it in the condensation assays. The syn-crDNA was labeled with 6-FAM at the 5′ end of the top strand. First, we confirmed the binding of HU protein to the syn-crDNA. As shown in Fig. 4B, HU displayed high affinity to the syn-crDNA with a Kd of 7.9 nM, which is comparable to the affinity of HU protein to its specific binding sites (27). We used the syn-crDNA for IPFM analysis with S9.6 antibody-coated beads in the presence of Cy-3– labeled naRNA4 by monitoring both 6-FAM and Cy-3 signals. Image merging showed that about 3% of the beads showed sig- nals, all of which were colocalized. Colocalization increased to Fig. 1. AFM analysis of naRNA4- and HU-mediated plasmid DNA conden- about 12% in the presence of HU (Fig. 4C and Table 1), sug- sation. For AFM images of supercoiled plasmid, pDNASC was present in every cr rel gesting that the syn- DNA can function as a target for naRNA4 experiment except in N and O, in which relaxed plasmid DNA, pDNA , was although the frequency of fluorescent beads to total counted used. The plasmid DNA (pDNA) is same plasmid, pSA508, that was used in the previous study (12). (A)pDNASC only. (B) +HU. (C) +naRNA4. (D) +HU+naRNA4. beads is somewhat less than that for supercoiled DNA (12 vs. (E) +naRNA4flat.(F) +HU+naRNA4flat.(G) +naRNA4YY.(H) +HU+naRNA4YY. 20%; Table 1), perhaps because of the restricted conforma- ZZ ZZ ZY ZY cr (I) +naRNA4 .(J) +HU+naRNA4 .(K) +naRNA4 .(L) +HU+naRNA4 . tional dynamics of the syn- DNA. Interestingly, when the (M)SameasinD but treated with RNaseH before AFM analysis. (N)pDNArel naRNA4 variants were applied, the frequency of DNA–RNA only. (O) +HU+naRNA4. (Scale bar: 100 nm.) complex formation changed compared with wild-type naRNA4

12226 | www.pnas.org/cgi/doi/10.1073/pnas.1711285114 Qian et al. Downloaded by guest on October 2, 2021 Fig. 2. IP-PCR analysis of plasmid DNA (pSA508) condensation by HU and naRNA4. Details are shown in Fig. S3A and Materials and Methods. M: DNA ladder (100 bp, from Fermentas). NC and PC are negative and positive controls for PCR, respectively. RNH is short for RNaseH. S and E fractions are supernatantsand eluates, respectively, during immunoprecipitation process using S9.6 antibody. After IP, DNA was extracted and purified from S and E, followed by PCR analysis using primers targeted to pSA508. The PCR products were separated by 1.5% agarose gel in 1× Tris-acetate-EDTA buffer.

(Table 1). The frequency decreased when naRNA4YY was used HU Acts as a Chaperone in naRNA4-Dependent DNA Condensation. As but increased in the case of naRNA4ZZ (8.2 and 15.6%, re- mentioned above, HU appears to be dispensable for naRNA4- spectively). However, when plasmid DNA was used, there was mediated DNA–DNA contacts but enhances the condensation no significant difference in the frequency in naRNA4 and its process (Figs. 3 and 4C and Table 1). Is it possible that HU plays variants (Table 1). a catalytic role (like a chaperone) in the DNA condensation GENETICS

Fig. 3. IP-FM analysis of naRNA4- and HU-mediated DNA condensation using plasmid DNA. The components in different assays are in the margins. RNH is short for RNaseH. Details are in Fig. S3B and Materials and Methods. The fluorescent images were processed by ImageJ. The images are representative of three experiments. (Scale bar: 1 μm.)

Qian et al. PNAS | November 14, 2017 | vol. 114 | no. 46 | 12227 Downloaded by guest on October 2, 2021 Table 1. Frequency of DNA–RNA complex in absence or that HU protein is not present in DNA condensates. We con- presence of HU firmed this observation by IP/IP PCR assays. After formation of Reaction components −HU +HU the condensates, the solution was first incubated with HU antibody-coated beads. After separation, the beads were washed, pDNASC+naRNA4 8/156 (5.1%) 46/157 (29.3%) and the eluates (E) were saved while the supernatants (S) were pDNASC+naRNA4flat 0/37 (0.0%) 0/104 (0.0%) further incubated with beads coated with S9.6, resulting in new SC YY pDNA +naRNA4 5/91 (5.5%) 21/68 (30.9%) supernatants (S/S) and eluates (S/E). DNA was extracted from all SC ZZ pDNA +naRNA4 5/96 (5.2%) 22/71 (31.0%) of the solutions and finally analyzed by PCR. We obtained signals SC ZY pDNA +naRNA4 3/64 (4.7%) 16/81 (19.8%) only from E when both HU and plasmid DNA were present, in- syn-crDNA+naRNA4 2/78 (2.6%) 11/92 (12.0%) dicating that HU binds to and saturates plasmid DNA (the mo- + YY syn-crDNA naRNA4 1/79 (1.3%) 7/85 (8.2%) lecular ratio of HU to DNA is 20:1 in the condensation assay). + ZZ syn-crDNA naRNA4 5/77 (6.5%) 12/77 (15.6%) When all three components were present, we did observe PCR + ZY syn-crDNA naRNA4 2/83 (2.4%) 10/81 (12.3%) signals from E and S/E, suggesting the formation of HU–DNA and Note: Frequency is defined as the ratio of beads with fluorescent signal to DNA–naRNA4 complexes, respectively (Fig. 5B). Furthermore, we total counted beads. performed IP/IP assays in combination with FM. After depleting HU and HU-bound complexes by beads with anti-HU antibody, the supernatants from IP were incubated with S9.6-coated beads, which process and is not present in the final condensate? To answer were subsequently washed and delivered to FM for imaging. We this question, we carried out IP–Western blot analysis of HU monitored the fluorescent signals from Cy-3–labeled naRNA4 in protein in the supernatants and eluates of IPP assays. Obviously, the beads using either plasmid DNA or cruciform DNA, while in HU was present only in the supernatants of the S9.6-coated thecaseofusing6-FAM–labeled syn-crDNA, we monitored both bead-bound complexes with both plasmid DNA and syn- Cy-3 and 6-FAM signals in the beads (Fig. 5C). Thus, we see that crDNA (Fig. 5A, lanes 5, 7, and 9), but was not detectable in the the final DNA–RNA complexes do not contain HU protein, which complex containing eluates (Fig. 5A, lanes 8 and 10), implying should have been immunoprecipitated by anti-HU antibody in the

Fig. 4. IP-FM analysis of naRNA4- and HU-mediated DNA condensation using syn-crDNA. (A) An expected secondary structure of syn-crDNA. The DNA was synthesized and labeled with 6-FAM at 5′.(B) Fluorescence polarization and HU binding to syn-crDNA. Different concentrations of HU protein (0–640 nM) were added to 1 nM 6-FAM–syn-crDNA. After incubation at room temperature for 10 min, fluorescence polarization (in units of mP) was obtained and plotted

as a function of HU concentration. Data analysis was carried out with Prism 7. Error bars show SD of triplicate experiments. The determined Kd is 7.9 nM. (C) Typical FM images. The components in different assays are in the margins. The fluorescent images were processed by ImageJ. The images are representative of three experiments. (Scale bar: 1 μm.)

12228 | www.pnas.org/cgi/doi/10.1073/pnas.1711285114 Qian et al. Downloaded by guest on October 2, 2021 Fig. 5. Confirmation of a chaperone role of HU in naRNA4/HU-mediated DNA condensation. (A) IP-Western blot analysis of HU in condensation complexes. M, Molecular weight protein markers. Different concentrations of HU (5, 10, 20, and 40 ng, lanes 1–4, respectively) were loaded as positive controls. S (lanes 5, 7, and 9) and E (lanes 6, 8, and 10) represent the supernatants and eluates from the beads, respectively. The components of the assays are labeled at the top.

(B) IP/IP-PCR analysis of plasmid DNA in condensation complexes. The reaction mixtures of condensation assays were incubated with anti-HU antibody, GENETICS resulting in supernatants (S) and eluates (E). Supernatants (S) were further incubated with S9.6 antibody, resulting in supernatants (S/S) and eluates (S/E). DNA was extracted and purified from the solutions and analyzed by 1.5% agarose gel after PCR using pSA508-targeted primers. (C) IP/IP FM analysis of Cy-3– naRNA4 and 6-FAM–syn-crDNA in condensation complexes. The components are in the left margins. Beads were washed with buffer three times before imaging. Images were processed by ImageJ. The images are representative of triplicate experiments. n/a, not applicable. (Scale bar: 1 μm.) (D) Schematic of HU-facilitated formation of DNA–RNA complex. HU protein (blue triangle) catalyzes DNA (green oval) and naRNA4 (yellow rectangle) to form the DNA–RNA complex and then dissociates from the complex. Theoretically, several modes of interaction between DNA and naRNA4 hairpins are possible (figure S1 and figure 5 in ref. 12). Among them are the formation of kissing DNA–RNA loops, parallel DNA–RNA heteroduplexes, Watson/Crick base pairing between some parts of the DNA and naRNA4 hairpin stems and hook-like connections between cruciform DNA loops and single-stranded naRNA4 loops. We are currently investigating the possible presence of any of these complexes in the DNA condensates.

previous step. These results strongly indicate that HU acts as a structures. The process involves DNA–RNA complex formation chaperone in the naRNA4-dependent DNA condensation. by HU. After facilitating formation of a DNA–RNA complex, HU protein dissociates from the complex (Fig. 5D). We propose Nature of DNA–RNA Complexes. The DNA–RNA complexes formed that HU proteins bind to both cruciform DNA and RNA hairpin in the DNA condensation (i) are sensitive to RNaseH and and bring them together, leading to the formation of DNA– (ii) bind to antibody against DNA–RNA hybrid, indicating the naRNA4 complexes. In the absence of HU proteins, the presence of some DNA–RNA hybrids. However, the fact that DNA–naRNA4 complexes are formed at a lower frequency, primary sequence alterations in several naRNA4 variants do not probably through spontaneous dynamic opening of cruciform lead to a loss of function suggests that there may not be extensive DNA structures (28, 29). Watson/Crick base pairing involved in the DNA–RNA complex How two such DNA–RNA complexes ultimately establish the formation. The ability of naRNA4 to target a variety of palin- DNA–DNA linkage remains unknown. The putative DNA–RNA dromic sequences also speaks against extensive DNA–RNA hy- intermediates do not necessarily have long stretches of Watson/ brid formation. In fact, only 2- to 4-bp-long stretches of Watson/ Crick base pairing. We are currently analyzing the structural Crick base pairing between naRNA4 and the syn-crDNA were characteristics of these DNA–RNA interactions. found by computer-based sequence analysis. The presence of such Role of noncoding RNA in various aspects of eukaryotic chro- a type of limited Watson/Crick base pairings may be sufficient for matinstructureandfunctionisubiquitous(reviewedinref.30).A recognition of the complexes by RNaseH and S9.6 antibody. few examples are heterochromatin assembly (31–33), gene silencing However, we cannot rule out other models of DNA–RNA com- (34), and X-inactivation (35, 36). Frequently, these RNAs are plex formation. We need to further investigate them. found to be located only in the segments of the chromosomes al- though it is not known how these RNAs interact with DNA. In case Discussion of lncRNA modified gene regulation, it has been shown that RNA We have shown that, in the presence of HU, naRNA4 helps does not interact with DNA (37, 38). Our findings—formation of DNA condensation by establishing contacts with cruciform DNA putative DNA–RNA complexes with limited Watson/Crick base

Qian et al. PNAS | November 14, 2017 | vol. 114 | no. 46 | 12229 Downloaded by guest on October 2, 2021 pairings and a chaperone role of HU in promoting the DNA–RNA ACKNOWLEDGMENTS. We thank Dr. Jiji Chen (Advanced Imaging & Micros- interactions during chromosome condensation in bacteria—are of copy Resource, NIH) for help in FM analysis; Dr. Emilios Dimitriadis much interest in chromatin research from mechanistic viewpoints. (Biomedical Engineering and Physical Science, NIH) for help with AFM; Dr. Stephen Leppla (National Institute of Allergy and Infectious Diseases, NIH) for providing the S9.6 antibody; and Dr. Robert Crouch (National In- Materials and Methods stitute of Child Health and Human Development, NIH) for the gift of RNase Procedures for RNA in vitro synthesis, DNA condensation, and immunoprecip- H. This work is supported by the Intramural Research Program of the Na- itation are described in SI Materials and Methods. Protocols for AFM, FM, and tional Institutes of Health, the National Cancer Institute, and the Center for fluorescence polarization (FP) are also provided in SI Materials and Methods. Cancer Research.

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