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Rapid emergence of evasive SARS-CoV-2 variants is an on-going challenge for COVID-19 vaccinology. Traditional virus neutralization tests provide detailed datasets of neutralization titers against the viral variants. Such datasets are difficult to interpret and do not immediately inform on the sufficiency of the breadth of antibody response. Some of these issues could be tackled using the antigenic cartography approach. In this study, we created antigenic maps using neutralization titers of sera from donors who received Sputnik V booster vaccine after primary Sputnik V vaccination and compared them with the antigenic maps based on serum neutralization titers of Comirnaty-boosted donors. Traditional analysis of neutralization titers against WT, Alpha, Beta, Delta, Omicron BA.1 and BA.4/BA.5 showed a significant booster humoral response after both homologous (Sputnik V) and heterologous (Comirnaty) revaccinations against all the studied viral variants. However, despite this, a more in-depth analysis using antigenic cartography revealed that Omicron variants remain antigenically distant from WT, which is indicative of the formation of insufficient levels of cross-neutralizing antibodies. The implications of these findings may be significant when developing a new vaccine regimen.

Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.

Context Progress achieved in the treatment of prostate cancer (PCa) with surgical, radiation, and hormonal therapies has drastically reduced mortality from this disease. Yet, patients with advanced PCa have few, if any, curative options. Recent success in treating patients with hematological malignancies of B-cell origin using T cells engineered to express chimeric antigen receptors (CARs) has inspired multiple groups worldwide to adapt this approach to the problem of late-stage PCa. Objective To summarize the available clinical results for CAR T-cell therapy of PCa and discuss future technological advancements in the CAR T-cell field that may help patients with metastatic PCa. Evidence acquisition A literature review was conducted of clinical trial data, abstracts presented at recent oncology conferences, as well as reports highlighting critical bottlenecks of CAR T-cell therapy that became apparent from preclinical and clinical studies. Evidence synthesis Current understanding of why CAR T-cell therapy may fail, particularly in the context of solid cancers, is as follows. First, a CAR design that provides potent activity and persistence of engineered T cells in the hostile tumor microenvironment is a must. The choice of the targetable epitope(s) is critical to counteract tumor antigen escape. Preclinical and clinical evidence indicates that the efficacy of CAR T-cell therapy can be enhanced significantly in combination with other therapeutic approaches. We propose that several improvements to CAR design and patient conditioning, such as unbiased identification of novel PCa-specific CAR targets, use of next-generation (multispecific, resistant to the tumor microenvironment, and with prolonged persistence) CAR T-cell products, and combination therapies may translate into improved patient outcomes and more durable responses. Conclusions Although significant preclinical experience of testing CAR T cells in solid cancer models has identified important technological and biological bottlenecks, information from clinical trials, particularly those focusing on the PCa, will be instrumental to the rational design of advanced CAR T therapies that will be both safe and effective in patients with advanced PCa. Patient summary So far, chimeric antigen receptor (CAR) T-cell therapy has not shown significant activity in patients with metastatic prostate cancer (PCa). CAR T-cell products used for such trials represent one of the pioneering efforts to adapt this technology to the problem of metastatic PCa. In retrospect, both CAR design and cell composition appear to have been suboptimal to expect strong patient responses. Given the impressive results of CAR-based approaches observed in preclinical models of solid cancers, emerging CAR T-cell products are expected to be more successful in the clinic. Here, we discuss the challenges that need to be overcome to boost the efficacy of PCa-targeted CAR T-cell therapy and call for dialogue between clinicians and cell biologists to address these challenges.

Adoptive T- and NK-cells transfer with chimeric antigenic receptors (CAR) is considered as a promising anticancer strategy. Chimeric antigenic receptors are artificial molecules that provide activation of the cells carrying them when they contact with a specific antigen to induce cell death. Unlike T cells, NK cells have no T-cell receptors, which prevent "graft-versus-host” disease under allograft transplantation. The aim of this work was to study antimetastatic activity of a double-modified human NK cell line YT - Cyto-CAR-YT-Lact cells carrying CAR to the PSMA protein and expressing antiapoptotic protein lactaptin. The BrCCh4e-134 breast carcinoma line with high PSMA expression was constructed by lentiviral transduction. The YT double modified NK cell line, Cyto-CAR-YT-Lact, expressing functional anti-PSMA CAR and carrying a deletion of the Shp-2 gene encoding the Shp-2 protein, a negative regulator of NK cell activation, was used as effector cells. The cytotoxic activity of Cyto-CAR-YT-Lact cells was tested on PSMA-positive BrCCh4e-134 cells and on parental BrCCh4e cell in vitro in real-time mode. The antimetastasis activity of Cyto-CAR-YT-Lact cells was analyzed in SCID and NOD/SCID mice with spontaneously metastases and intravenously transplanted PSMA-positive BrCCh4e-134 cells. Cyto-CAR-YT-Lact cells efficiently reduced the viability of PSMA-positive tumor cells and moderately PSMA-negative target cells in vitro. As a tumor model, we used human breast cancer cells that overexpress PSMA as a transgene and are characterized by metastasis to the mediastinal lymph nodes being transplanted on mice. It was shown that a single intravenous administration of the Cyto-CAR-YT-Lact cells suppressed the development of metastases in the spontaneous metastasis model and when tumor cells were introduced into the bloodstream. The reaction of the primary tumor node to Cyto-CAR-YT-Lact therapy was not detected. Thus, the use of modified NK cells can be considered as a promising therapeutic approach for suppressing metastasis, but not for suppressing the growth of the primary tumor node.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2. Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. In vitro stimulated MBCs from previously infected recipients of Sputnik V secreted a significant amount of anti-RBD IgG both on days 28 and 85 from the beginning of vaccination. These antibodies demonstrated robust neutralization of the Wuhan Spike-pseudotyped lentivirus. In the naïve group of vaccinees, the level of anti-RBD IgG secretion was five- to six-fold reduced compared to that of the recovered group, and maximum virus neutralization (Wuhan spike) was achieved only on day 85. Sera from all the recovered and most naïve Sputnik V recipients were neutralizing against the ancestral Wuhan and mutant B.1.351 viruses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

Cloning VH and VL genes from individual antigen-specific B cells is an attractive approach for producing monoclonal antibodies of the desired specificity. Current RT-PCR protocols, however, result in the successful identification of VH and VL gene pairs in about half of the sorted cells. Here, we demonstrate that single-cell RT-PCR is likely affected by stochastic factors, and that running PCRs in triplicate results in successful amplification of the expressed VH and VL genes in 90–100% of single sorted human B cells.

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

Background: The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, several key immunological features of Sputnik V, an adenovirus-based two-component vaccine against SARS-CoV-2, need to be explored in detail. These include the effects of Sputnik V on B cell immunity and its ability to elicit antibody responses that are active against emerging neutralization-resistant SARS-CoV-2 variants. Methods: Our study included a cohort of 22 volunteers who received complete Sputnik V vaccination (two doses 21 days apart), 5 of who had a recent history of mild COVID-19, and 17 constituted a group of SARS-CoV-2 unexposed individuals. The frequencies of receptor-binding domain (RBD)-specific plasmablasts and B memory cells (MBCs), circulating and MBC-derived antibody secreting cells, virus binding, and virus-neutralizing activities of the sera, as well as samples of MBC-derived antibodies were analyzed using flow cytometry, ELISA, enzyme-linked immunosorbent spot (ELISpot), and pseudotyped virus neutralization assay at four time points spanning the period immediately before, during, and after vaccination. Findings: Longitudinal analysis of circulating serum antibodies showed that the anti-RBD IgG levels in naive vaccine recipients substantially increased after the second vaccine dose (P

Immunotherapy based on adoptive transfer of genetically engineered T- and NK-cells is an area of active ongoing research and has proven highly efficacious for patients with certain B-cell malignancies. Use of NK cells and NK cell lines as carriers of chimeric antigen receptors (CARs) appears particularly promising, as this opens an opportunity for moving the therapy from autologous to the allogeneic (universal) format. This "off-the-shelf" approach is thought to significantly reduce the price of the treatment and make it available to many more patients in need. Yet, the efficacy of CAR-NK cells in vivo presently remains low, and boosting the activity of CAR NK cells via stronger tumor homing, resistance to tumor microenvironment, as well as greater cytotoxicity may translate into improved patient outcomes. Here, we established a derivative of a human NK cell line YT overexpressing a positive regulator of cytotoxicity, VAV1. Activity of YT-VAV1 cells obtained was assayed in vitro against several cancer cell lines and primary patient-derived cancer cells. YT-VAV1 cells outperform parental YT cells in terms of cytotoxicity.

T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs. Human Fn3 domains have lower predicted immunogenicity compared to mouse-derived sequences, and a reduced molecular weight compared to scFvs. We created a functional CAR using a VEGFR2-specific Fn3 module replacing the conventional scFv. The resulting FnCAR specifically potentiates the cytotoxic activity of human T cells and YT NK cells in the presence of VEGFR2-positive targets. These findings demonstrate that Fn3 domains can be used in CARs for antigen recognition.

Despite the multitude of anticancer cytostatic drugs available to oncologists today, most of such drugs have serious side effects that may preclude their use in some groups of patients. Hence, selective induction of apoptosis in cancer but not normal cells remains an attractive goal of molecular medicine. Lactaptin, a proteolytic fragment of the human milk kappa-casein, has been previously identified as a protein displaying potent killing of cancer cells in vitro. Its recombinant analog (RL2) produced in E. coli has been shown to delay solid tumor growth in vivo. Given that lactaptin is of human origin and is not immunogenic, it can be administered to patients multiple times without running the risk of immune response that could dampen the therapy efficacy. In the present study, we demonstrate that the combination of RL2 and cyclophosphamide treatments has an additive therapeutic effect against hepatoma tumor in immunocompetent mice. We asked whether production of lactaptin in human rather than bacterial cells would result in a protein with increased cytotoxic activity. Using lentiviral vector pCDH as a backbone, two constructs, pEL1 and pEL2, encoding secreted forms of lactaptin that differ in their signal sequences were created. Lactaptin expression in human cell lines was confirmed using Western-blot analysis, whereas ELISA was used for quantification of secreted lactaptin. Next, we measured the cytotoxic effects of the media conditioned by pEL1-transfected HEK293T cells, as assayed against the panel of three human cancer cell lines: MDA-MB-231 (adenocarcinoma), PC3 (prostate cancer), and T98G (glioblastoma). We show that EL1-derived lactaptin is at least 100-fold more cytotoxic than RL2. Taken together, our results provide an opportunity for developing armored immune cells as an “off-the-shelf” platform for targeted delivery of lactaptin to cancer cells.

The adoptive transfer of allogeneic CAR NK cells holds great promise as an anticancer modality due to the relative ease of manufacturing and genetic modification of NK cells, which translates into affordable pricing. Compared to the pronounced efficacy of CAR T cell therapy in the treatment of B cell malignancies, rigorous clinical and preclinical assessment of the antitumor properties of CAR NK cells has been lagging behind. In this brief review, we summarize the biological features of NK cells that may help define the therapeutic niche of CAR NK cells as well as create more potent NK cell-based anticancer products. In addition, we compare T cells and NK cells as the carriers of CARs using the data of single-cell transcriptomic analysis.

Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering.

Early results from clinical trials of autologous chimeric antigen receptor (CAR)-expressing T cells for the therapy of B-cell malignancies have encouraged extending the potency of this therapy to other cancers. However, the success of using CAR T-cells to treat patients with solid tumors has been limited. In this review, we summarize current knowledge on the design and applications of CARs for the targeted therapy of cancer. We describe existing issues that limit the widespread application of CAR T cells and discuss the optimization steps needed to further improve safety and efficacy of this therapeutic platform.

The article reports the development of a collection of lentiviral vector constructs enabling time-efficient production and testing of different variants of chimeric antigen receptors (CAR). These artificial surface proteins make it possible to redirect the activity of immune cytotoxic T-cells towards cancer cells. Chimeric antigen receptors usually encompass four functional modules, namely, antigen recognition, flexible linker, transmembrane, and signal modules. The use of modules with different properties allows modulating the affinity and specificity of CAR interaction with target antigens, as well as intensity and quality of activation signaling, which determines the cytotoxic properties of CAR T-cells, as well as their proliferation rate and time of persistence in the organism. The proposed vector system make it possible to easily test various combinations of CAR modules while its being open to distribution allows the direct comparison of the results obtained by different scientific groups.

Multiple lines of evidence indicate that CAR-T cell based therapy and oncolytic virotherapy display robust performance in both immunocompetent and immunodeficient mouse models. Rare, yet highly successful attempts to combine these therapeutic platforms have also been reported. Interestingly, both approaches have shown pronounced efficacy in human trials, albeit these were limited to just a handful of malignancies. Specifically, CD19-specific CAR-T cell products (Kymriah and Yescarta) have been highly effective against B cell lymphomas and leukemias, whereas administering oncolytic viruses resulted in pronounced responses in melanoma (Imlygic and Rigvir) and nasopharyngeal carcinoma (Oncorine) patients. It is well established that efficacy of virotherapy as a standalone approach is largely restricted by the pre-existing and mounting immune response against viral antigens, and requires a relatively functional immune system, which is not typical for cancer patients, with the current antitumor therapy schemes. On the other hand, the most important challenges faced by the current CAR-T cell therapy formats include the lack of targetable tumor-specific surface antigens, tumor cell heterogeneity, and immunosuppressive tumor microenvironment, not to mention the unacceptably high costs. Remarkably, combining the two approaches may help address their individual bottlenecks. Namely, local acute inflammatory reaction induced by the viral infection may reverse tumor-associated immunosuppression and lead to more efficient homing and penetration of CAR-expressing lymphocytes into the tumor stroma; combined viral and CAR-mediated cytotoxicity may ensure the production of immunogenic cell debris and efficient presentation of tumor neoantigens, and potently recruit the patient's own bystander immune cells to attack cancer cells. Thus, testing the combinations of CAR-based and virolytic approaches in the clinical setting appears both logical and highly promising.

Background Cytotoxic activity of T- and NK-cells can be efficiently retargeted against cancer cells using chimeric antigen receptors (CARs) and rTCRs. In the context of solid cancers, use of armored CAR T- and NK cells secreting additional anti-cancer molecules such as cytokines, chemokines, antibodies, BiTEs, inverted cytokine receptors, and checkpoint inhibitors, appears particularly promising, as this may help overcome immunosuppressive tumor microenvironment, attract bystander immune cells, and boost CAR T/NK-cell persistence. Placing the expression of such molecules under the transcriptional control downstream of CAR-mediated T/NK-cell activation offers the advantage of targeted delivery, high local concentration, and reduced toxicity. Several canonic DNA sequences that are known to function as activation-inducible promoters in human T and B cells have been described to date and typically encompass the multimers of NFkB and NFAT binding sites. However, relatively little is known about the DNA sequences that may function as activation-driven switches in the context of NK cells. We set out to compare the functionality of several activation-inducible promoters in primary human T cells, as well as in NK cell lines NK-92 and YT. Methods Lentiviral constructs were engineered to express two fluorescent reporters: mCherry under 4xNFAT, 2xNFkB, 5xNFkB, 10xNFkB, 30xNFkB promoters, as well as two variants of the CD69 promoter, and copGFP under the strong constitutive promoter of the human EF1a gene. Pseudotyped lentiviral particles obtained using these constructs were transduced into primary human T cells and NK-92 and YT cell lines expressing a CAR specific for PSMA. The transgenic cells obtained were activated by CD3/CD28 beads (T cells) or via a CAR (CAR-NK cell lines). Promoter activity before and after activation was assayed using FACS analysis. Results In T cells, the CD69 promoter encompassing CNS1 and CNS2 regions displayed the highest signal/noise ratio. Intriguingly, in the context of CAR-YT cell line neither of the seven promoters tested displayed acceptable activation profile. In CAR-NK-92 cells, the largest fold activation (which was modest) was achieved with the 10xNFkB and 30xNFkB promoters, however its expression was clearly leaky in “resting” non-activated cells. Conclusions Unlike in T cells, the robust activation-driven inducible expression of genetic cassettes in NK cells requires unbiased genome-wide identification of promoter sequences. Electronic supplementary material The online version of this article (10.1186/s12920-019-0489-4) contains supplementary material, which is available to authorized users.

Significance The posttranslational modification of nucleosomes is implicated in the regulation of gene expression and chromatin packaging in all eukaryotes. In this study, we investigate the function of histone H4 lysine 16 (H4K16) and its acetylation in Drosophila by generating strains in which lysine 16 is mutated to arginine, glutamine, or alanine. The main conclusion of our paper is that even though H4K16 acetylation was reported to be a critical regulator of chromatin folding in vitro and has therefore been assumed to affect many different nuclear processes, its essential function in Drosophila is in one process: X-chromosome dosage compensation in males., Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome–nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. Here, we analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription.

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein–protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.

The MSL complex acts on chromatin to and is required for X-chromosome dosage compensation. Chromatin-interacting protein MS (ChIP-MS) is now used to identify proteins and histone modifications interacting with the MSL complex, leading to the identification of CG4747. Functional analysis indicates that this protein is involved in targeting MSL to H3K36me3-containing chromatin.

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function., United States. Dept. of Energy (Contract DE-AC02-05CH11231), RC2 HG005639, U01 HG004279, R01 GM082798, R37 GM45744, R01 GM071923, U54 HG004592, National Science Foundation (U.S.) (NSF 0905968)

For the dosage compensation to occur, genes on the single male X chromosomes in Drosophila must be selectively bound and acetylated by the ribonucleoprotein complex called MSL complex. It remained unknown how such exquisite specificity is achieved, and whether specific DNA sequences were involved. In the present work we demonstrate that it is transcription of the gene on the X chromosome that is important for MSL targeting, irrespective of gene origin and DNA sequence.

The Drosophila MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16 and increase expression approximately 2-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We hypothesized that sequence-specific targeting occurs at "chromatin entry sites," but the majority of sites are sequence independent. Here we characterize 150 potential entry sites by ChIP-chip and ChIP-seq and discover a GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome ( approximately 2-fold), but this is doubled when considering its preferential location within or 3' to active genes (>4-fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.

We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.

Using electron microscopic (EM) data on the formation of a novel band from theP-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, 3C5-6/C7, and 60E8-9/E10 of Drosophila melanogasterpolytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequence removed by deletion Df(1)fa swb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophilagenome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5" and 3" nontranslated gene regions are located. These results suggest that Drosophilainterbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.

Dosage compensation has arisen in response to the evolution of distinct male (XY) and female (XX) karyotypes. In Drosophila melanogaster, the MSL complex increases male X transcription approximately twofold. X-specific targeting is thought to occur through sequence-dependent binding to chromatin entry sites (CESs), followed by spreading in cis to active genes. We tested this model by asking how newly evolving sex chromosome arms in Drosophila miranda acquired dosage compensation. We found evidence for the creation of new CESs, with the analogous sequence and spacing as in D. melanogaster, providing strong support for the spreading model in the establishment of dosage compensation.

This study shows how young sex chromosomes have altered their chromatin structure in Drosophila, and what genomic changes have led to silencing of the Y, and hyper-transcription of the X., Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome., Author Summary Sex chromosomes differ from non-sex chromosomes (“autosomes”) at the genomic, transcriptomic, and epigenomic level, yet the X and Y share a common evolutionary origin. The Drosophila Y chromosome is gene-poor and associated with a compact and transcriptionally inactive form of genetic material called heterochromatin. The X, in contrast, is enriched for activating chromatin marks and is consequently hyper-transcribed, a process thought to be an adaptation to decay and silencing of genes on the Y, resulting in “dosage compensation.” How sex chromosomes have altered their chromatin structure, and what genomic changes led to this dramatically different epigenetic makeup, however, has remained a mystery. By studying the genome, epigenome, and transcriptome of a species with a very recently evolved pair of sex chromosomes (the neo-X and neo-Y of a fruit fly, Drosophila miranda), we here recapitulate how both dosage compensation and heterochromatin formation evolve in Drosophila and establish several novel and important principles governing the evolution of chromatin structure. We dissect the evolutionary history of over 60 novel binding sites for the dosage compensation complex that evolved by natural selection on the neo-X within the last one million years. We show that the 21-bp consensus motifs for recruiting the dosage compensation complex were acquired by diverse molecular mechanisms along the neo-X, while the onset of heterochromatin formation is triggered by the accumulation of transposable elements, leading to silencing of adjacent neo-Y genes. We find that spreading of these chromatin modifications results in massive mis-expression of neo-sex linked genes, and that little correspondence exists between functional activity of genes on the neo-Y and whether they are dosage-compensated on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those that are heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities of the ancestral chromosome that formed the sex chromosome to evolve active versus repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution.

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome., Author Summary The genomes of complex organisms encompass hundreds of millions of base pairs of DNA, and regulatory molecules must distinguish specific targets within this vast landscape. In general, regulatory factors find target genes through sequence-specific interactions with the underlying DNA. However, sequence-specific factors typically bind only a fraction of the candidate genomic regions containing their specific target sequence motif. Here we identify potential roles for chromatin environment and flanking sequence composition in helping regulatory factors find their appropriate binding sites, using targeting of the Drosophila dosage compensation complex as a model. The initial stage of dosage compensation involves binding of the Male Specific Lethal (MSL) complex to a sequence motif called the MSL recognition element [1]. Using data from a large chromatin mapping effort (the modENCODE project), we successfully identify an active chromatin environment as predictive of selective MRE binding by the MSL complex. Our study provides a framework for using genome-wide datasets to analyze and predict functional protein–DNA binding site selection.

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the “silencing” marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin–heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both “activation” marks (e.g., H3K4me3 and H3K36me3) and “silencing” marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

Dosage compensation in Drosophila melanogaster males is achieved via targeting of male-specific lethal (MSL) complex to X-linked genes. This is proposed to involve sequence-specific recognition of the X at ∼150–300 chromatin entry sites, and subsequent spreading to active genes. Here we ask whether the spreading step requires transcription and is sequence-independent. We find that MSL complex binds, acetylates, and up-regulates autosomal genes inserted on X, but only if transcriptionally active. We conclude that a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL binding. Instead, linkage and transcription play the pivotal roles in MSL targeting irrespective of gene origin and DNA sequence.