Decoding chromatin states by proteomic profiling of nucleosome readers.

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens.

The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.

Management of macula-on giant retinal tear detachments- outcome of pars-plana-vitrectomy with silicone oil versus gas tamponade.

BACKGROUND: To compare the outcome of eyes with a macula-on giant retinal tear (GRT) detachment treated with pars-plana-vitrectomy (PPV) depending on the used endotamponade. METHODS: All consecutive cases with a macula-on GRT-associated rhegmatogenous retinal detachment (RRD) managed with PPV between 2007 and 2022 were retrospectively assessed depending on the selected endotamponade. By reviewing medical charts and surgical protocols the pre- and intraoperative parameters were analysed in detail. The number of vitreoretinal (VR) procedures needed for reattachment, the redetachment rate and the functional outcome were evaluated. Eyes treated with primary silicone oil (SO) tamponade were compared to eyes with primary gas tamponade. Cases with pre-existing conditions affecting outcome e.g. macula-off situation, history of trauma, status after complicated cataract surgery, former VR surgery or proliferative vitreoretinopathy grade C or higher were excluded. RESULTS: Overall, 51 eyes of 45 patients with a macula-on GRT detachment were treated with PPV and SO (n = 32; 63%) or gas (n = 19; 37%) endotamponade in the observed period. Eyes with primary SO tamponade underwent on average 2.3 (SD 0.8) VR procedures and had a redetachment rate of 13% (n = 4). Eyes with gas tamponade showed a higher redetachment rate of 32% (n = 6) with a mean number of 1.6 (SD 1.0) PPV procedures. Postoperative best-corrected visual acuity (BCVA) was significantly better in eyes with primary gas tamponade (mean logMAR BCVA 0.32; SD 0.30) compared to eyes with SO (mean logMAR BCVA 0.60; SD 0.42; p = 0.008). CONCLUSIONS: Surgical management of GRT-associated RRDs is complex. In clinical routine often SO is used as endotamponade. Because of known disadvantages (second procedure necessary for SO removal, unexplained SO-related visual loss, secondary glaucoma, SO emulsification) some VR surgeons prefer a gas tamponade. In our cohort, eyes with a gas compared to SO tamponade showed higher redetachment rates. However, the final postoperative BCVA was significantly better in eyes with gas compared to SO tamponade. TRIAL REGISTRATION: The trial protocol was approved by the local ethics committee on 25th of November 2022 (Ethikkommission der Universität Regensburg, Votum 22-3166-104).

Characteristics of Bilateral Retinal Detachment.

INTRODUCTION: Rhegmatogenous retinal detachment (RD) is still a sight-threatening and potentially blinding disease, especially if both eyes are affected. The purpose of this study is analysing the specific characteristics of bilateral rhegmatogenous RD. METHODS: The files of all 5,791 consecutive eyes undergoing vitreoretinal surgery for uncomplicated RD in a single tertiary retinal centre between January 2005 and June 2021 were retrospectively reviewed. RESULTS: A total of 300 patients (600 eyes) had bilateral retinal detachment. Interval between initial and subsequent RD surgery was 2.6 ± 2.8 (mean ± SD, median 1.5) years. Symptoms were reported by the patients for 20 ± 75 (median 5) days before presentation in the initial eye and 12 ± 32 (median 4) days in the subsequent eye. 220 patients were male (73%), and mean age at initial RD was 55 years. 183 (61%) of the initial RD eyes were phakic. In the initial eye, more patients had a detached macula, worse visual acuity, and more quadrants involved. Primary anatomic success rate was higher in the subsequent eye (90%) compared to the initial eye (83%). There was no difference in the reattachment rate of fellow eyes with primary failure in the first eye (91%) compared to those with primary success in the first eye (90%). There was a high symmetry between the eyes in terms of type of retinal break, number of breaks, and presumed localization of the causative retinal break. CONCLUSION: Patients with bilateral RD were more commonly male and younger than the group of all RD patients. The proportion of pseudophakia was not different. The majority of fellow eye RD occurred within 2 years after the RD in the first eye. Second eye RD was less advanced and had a better anatomical repair rate. Despite their experience in the first eye and despite typical symptoms, patients presented only after a mean of 12 days with RD in the second eye. RD in the initial and the subsequent eye showed a high symmetry. The anatomic result in the first eye is not a predictor for the anatomic result in the subsequent eye.

Two distinct modes for propagation of histone PTMs across the cell cycle.

Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how histone marks propagate and demonstrate that chromatin states oscillate within the cell cycle.

Are Retinal Detachments Becoming More Frequent? Data from 2 University Eye Clinics and Literature Review. / Werden Netzhautablösungen häufiger? Daten von 2 Universitätsaugenkliniken und Literaturübersicht.

There have been marked increases in the numbers of patients with retinal detachments at individual centres in recent years and this is supported by the subjective impression of many experts. We therefore surveyed the literature on changes in the incidence of retinal detachments worldwide. This revealed quite significant methodological differences between the studies, so that it was difficult to achieve a conclusive comparison of the development of the incidence of retinal detachment. Despite these limitations, all data from recent studies suggest an increase in the number of retinal detachments. The incidence of retinal detachment in the western world currently seems to be more than 20 cases per 100,000 person-years, which is significantly higher than described in earlier decades. It can be assumed that an increase in the number of individuals with myopia, a demographic increase in patients of the typical age group for retinal detachment and an increasing number of cataract surgeries, especially in younger patients, are responsible for the rising incidence of retinal detachment.

Current Practice in the Treatment of Epithelial and Melanocytic Tumours with Interferon-α2b: A Survey of Tertiary Eye Centres in Germany.

PURPOSE: To evaluate the standard of care, in particular the use of topical or subconjunctival interferon-α2b, in treating ocular surface squamous neoplasia or melanocytic tumours in tertiary eye centres in Germany. METHODS: A survey containing 14 questions was sent to 43 tertiary eye centres in Germany. The questions addressed the surgical and medical management of ocular surface squamous neoplasia and melanocytic tumours (primary acquired melanosis and malignant melanoma), as well as the clinical experiences and difficulties in prescribing off-label interferon-α2b eye drops and subconjunctival injections. RESULTS: Twenty-four tertiary eye centres responded to the survey. Eighty-three percent of centres had used interferon-α2b in their clinical practice and 25% prescribed it as the first-line cytostatic agent following surgical excision of ocular surface squamous neoplasia, while 10% would do so for melanocytic tumours. Correspondingly, the majority of respondents selected mitomycin C as their first-line agent. Side effects were uncommon with topical interferon-α2b eye drops but were more frequently reported after subconjunctival interferon-α2b injections. In total, eight centres had experience with interferon-α2b injections. The most significant obstacles perceived by ophthalmologists when prescribing interferon-α2b were its high cost and the reimbursement thereof. CONCLUSION: Off-label mitomycin C was the preferred adjuvant therapy for epithelial and melanocytic tumours, with interferon-α2b being the standard second-line option. Interferon-α2b has predominantly been used to treat ocular surface squamous neoplasia and, to a lesser extent, melanocytic tumours at German tertiary eye centres. Following its market withdrawal, supply shortages of interferon-α2b are likely to have a profound impact on patient care and their quality of life.

Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.

Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.

Redundant mechanisms to form silent chromatin at pericentromeric regions rely on BEND3 and DNA methylation.

Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin.

Rhegmatogenous retinal detachment repair-does age, sex, and lens status make a difference?

PURPOSE: To analyze the correlation between lens status, age, and sex in the epidemiology and success rates of rhegmatogenous retinal detachment (RRD) surgery. METHODS: The files of all consecutive patients undergoing vitreoretinal surgery for uncomplicated RRD between Jan 2005 und Dec 2020 were retrospectively reviewed. Successful outcome was defined as no retinal redetachment occurring within 3 months after surgery. RESULTS: 5502 eyes with uncomplicated primary RRD were included. Mean age of the patients was 61.1 years (± 13.6 SD). In the age group over 40 years, a male predominance was found. The percentage of pseudophakic RRD increased from 25 to 40% during the 15 years observation period. In the age group 50 to 69 years, patients with pseudophakic detachments were male in 786 out of 1079 cases (72.9%). In the same age group, 1285 of 2110 (60.9%) patients with phakic RRD were male. Overall, primary success rate after one procedure was 91.2% (5018 of 5502). In the phakic eyes, the primary success rate was higher in those eyes that underwent combined phacovitrectomy (93.0%), compared to those without simultaneous cataract surgery (88.7%; p = 0.002). CONCLUSION: The ratio of male and female patients with RRD varies between age groups. The proportion of pseudophakic RRD has increased within 15 years. The male predominance in RRD is stronger in pseudophakic than in phakic eyes. In phakic eyes with RRD, a combined phacovitrectomy yielded better anatomical results.

The learning curve of retinal detachment surgery.

PURPOSE: To investigate the learning curve of vitreoretinal (VR) surgeons beginning training in retinal detachment (RD) surgery. METHODS: The files of all consecutive patients undergoing VR surgery for uncomplicated RD between Jan 2005 und Mar 2020 were retrospectively reviewed. Successful outcome was defined as no retinal redetachment within 3 months after surgery. RESULTS: Ten surgeons started their VR career during this period. Together, these 10 surgeons performed 3786 RD operations (mean 379; median 251; range 71-1053). Primary success rate after one operation was 90% (3420 of 3786). When starting to operate retinal detachments, VR surgeons had a primary success rate of about 80%. Redetachment rates steadily decreased and stabilized at just under 10% after about 200 operations. Beginners needed more than twice the time for the procedure compared to experienced surgeons. The individual learning curves varied widely. In our series, female surgeons seem to have a faster learning curve. CONCLUSION: RD surgery performed by VR surgeons in training had acceptable results. With increasing experience, success rates continuously improve reaching stable levels after approximately 200 operations. The training of VR surgeons requires considerable resources.

Ischemic Choroidal Diseases. / Ischämische Aderhauterkrankungen.

INTRODUCTION: Ischemic choroidal diseases are an underdiagnosed entity. The clinical pattern varies according to the size and the localisation of the affected vascular structure. CLINICAL PRESENTATION: In eyes with occlusion of the long posterior ciliary arteries, characteristic triangular patches of choroidal ischemia (Amalric sign) are seen, which in the course of time merge into well-defined areas of atrophy of the retinal pigment epithelium. Above the non-perfused choroidal areas, hyperpigmented, grouped lines appear (Siegrist streaks). Circumscribed ischemia of smaller choroidal arterioles and capillary vessels appears as multifocal, yellowish lesions in the posterior fundus (Elschnig spots). Vortex vein occlusion becomes manifest as exudative haemorrhagic choroidal swelling in the periphery. CAUSES OF CHOROIDAL ISCHEMIA: Apart from arterial hypertension as a major risk factor, some immunological disorders such as giant cell arteritis and systemic lupus erythematosus and haematological pathologies also affect choroidal perfusion. Furthermore, choroidal ischemia occurs due to local inflammation, as found in eyes with acute multifocal posterior placoid pigment epitheliopathy (APMPPE). Rarely, choroidal infarction is of iatrogenic origin or drug-induced. Recent advances in imaging, such as the introduction of enhanced depth imaging optical coherence tomography (EDI-OCT) and OCT angiography (OCT-A), have improved the visualisation of the choroidal vasculature and complement the classical angiographic procedures. In patients with age-related macular degeneration (AMD) and diabetes, some changes in choroidal blood flow and vascular structure have also been noted. While in AMD the choroidal pathologies correlate with the disease progression and the functional prognosis, the pathophysiological relationship between diabetic choroidopathy and retinopathy is currently unclear. MANAGEMENT AND CONCLUSION: With regard to the limited therapeutic options for choroidal ischemia, optimisation of the cardiovascular risk profile and the management of accompanying ocular and systemic diseases are essential.

[Diabetic retinopathy]. / Diabetische Retinopathie.

Diabetic retinopathy (DR) is a vision-threatening microvascular complication of diabetes and the leading cause of blindness in working-age people. At the beginning of the metabolic disorder and in early stages of DR the patient's eyesight is often not affected. Depending on the duration of diabetes and in more advanced stages of DR the vision is compromised through the presence of diabetic macular edema (DME) and/or proliferative retinal complications. The management of DR comprises regular ophthalmic examinations according to clinical guidelines, the targeted application of multimodal imaging, and the specific treatment of DME and proliferative DR including secondary disorders such as neovascular glaucoma or persistent vitreous haemorrhage. Innovative ocular imaging techniques like optical coherence tomography (OCT), OCT angiography (OCT-A) and ultrawide field imaging play an important role in the assessment of diabetic patients. Various non-invasive imaging modalities have become part of the routine clinical work-up and help to identify new biomarkers for early diagnosis and long-term prognosis. In early stages of DR, the multifactorial intervention including glucose level and blood pressure control as well as optimizing the patient's cardiovascular risk profile is essential. A specific ophthalmic therapy is available for DME and proliferative DR (PDR). In patients with PDR the treatment regime includes panretinal laser photocoagulation or alternatively intravitreal anti-VEGF (vascular endothelial growth factor)-injections accompanied by close-meshed clinical monitoring. In patients with both, DME and PDR, it is suggested to start with Anti-VEGF drugs. In severe PDR with persistent vitreous haemorrhage, tractional maculopathy or tractional retinal detachment vitreoretinal surgery is recommended.

[Diabetic Macular Edema]. / Diabetisches Makulaödem.

Diabetic macular edema (DME) is a chronic retinal disease, which requires intensive clinical monitoring. Within the last ten years the intravitreal anti-VEGF (vascular endothelial growth factor) therapy has become the standard of care to improve and stabilize vision in patients with centre involving DME. Long-acting intravitreal corticosteroids can achieve similar visual results with fewer injection rates. Because of steroid-induced side effects (progression of cataract, glaucoma) these drugs are regarded as second-line medication. Since the introduction of anti-VEGF-medication the focal laser photocoagulation is no longer considered as first-line therapy for DME. However, a focal laser treatment can sometimes be a possible alternative in specific situations. In patients with proliferative diabetic retinopathy and DME, the intravitreal anti-VEGF therapy is approved for both conditions. In ischemic maculopathy the functional outcome is restricted. For the indication of anti-VEGF-treatment for DME with accompanying central ischemia not only visual acuity and optical coherence tomography parameters should be considered, the amount of ischemia seen on fluorescein angiography should also be taken into account. In tractional macular edema due to epiretinal membranes and vitreomacular adhesions a pars-plana vitrectomy with membrane peeling is indicated.

Intravitreal anti-VEGF treatment for subretinal neovascularisation secondary to type 2 idiopathic juxtafoveolar telangiectasia.

PURPOSE: To assess the long-term outcome of patients with subretinal neovascular membrane (SRNVM) secondary to type 2 idiopathic juxtafoveolar telangiectasia (IJT) receiving intravitreal anti-VEGF (vascular endothelial growth factor) injections. METHODS: A total of 14 eyes of 12 patients treated with intravitreal anti-VEGF for SRNVM related to type 2 IJT were retrospectively assessed. RESULTS: Nine men and 3 women with a mean age of 66 years (SD 12, range 47-87 years) were diagnosed with IJT-related SRNVM. On average, 6.8 injections (SD 5.5, range 3-18) were given per eye. Ten eyes were treated with ranibizumab, 3 eyes with bevacizumab and 1 eye received both substances. The median follow-up after the last injection was 31 months (IQR: 18, 48). In 6 eyes, BCVA improved by 1-4 lines (mean Δ +2.0 lines), 1 eye remained stable and 7 eyes showed decline of vision by 1-5 lines (mean Δ -2.1 lines). The baseline central foveal thickness was significantly reduced from a mean of 323 (SD 87) to 266 µm (SD 71 µm) at the last follow-up visit (p = 0.001). CONCLUSIONS: SRNVM development is a severe complication of type 2 IJT. Since the establishment of intravitreal anti-VEGF treatment laser coagulation and PDT have lost significance. Intravitreal anti-VEGF therapy seems to be safe and effective for the treatment of IJT-related SRNVM. Frequently multiple intravitreal injections are necessary for stabilisation.

Clinical Features and Outcome of Paediatric Retinal Detachment.

PURPOSE: The aim of this study was to assess the clinical features and outcome of paediatric retinal detachment (RD). METHODS: Ninety-five eyes of 87 children under 18 years of age with RD were assessed. The risk factors, morphology of RD, therapeutic approach and functional results were evaluated. RESULTS: Sixty-seven boys and 20 girls with a mean age of 10.4 years (standard deviation 5.5) presented with RD. The following risk factors were identified: myopia (23%), congenital or developmental ocular abnormalities (37%), history of ocular trauma (40%) and previous ocular surgery (27%). Seventy-seven (81%) eyes underwent surgery. The primary reattachment rate was 44%. In 18%, reattachment was achieved after several surgeries. The overall recurrence rate after surgical reattachment was 39%. CONCLUSIONS: Paediatric RD is a sight-threatening condition. Often, aggravating factors, such as delayed diagnosis, hereditary ocular abnormalities or strong vitreous adherence, are present. Hopefully, modern surgical techniques may contribute to a better outcome of RD in the future.

Circadian control of fatty acid elongation by SIRT1 protein-mediated deacetylation of acetyl-coenzyme A synthetase 1.

The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD(+)-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

Fast signals and slow marks: the dynamics of histone modifications.

Most multi-cellular organisms adopt a specific gene expression pattern during cellular differentiation. Once established, this pattern is frequently maintained over several cell divisions despite the fact that the initiating signal is no longer present. Differential packaging into chromatin is one such mechanism that allows fixation of transcriptional activity. Recent genome-wide studies demonstrate that actively transcribed regions are characterized by a specific modification pattern of histones, the main protein component of chromatin. These findings support the hypothesis that a histone code uses histone post-translational modifications to stably inscribe particular chromatin structures into the genome. Experiments on the dynamics of histone modifications reveal a striking kinetic difference between methylation, phosphorylation and acetylation, suggesting different roles of these modifications in epigenetically fixing specific gene expression patterns.

Identification of novel Drosophila centromere-associated proteins.

Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).