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Aims: Dynamic alterations in cardiac DNA methylation have been implicated in the development of heart failure (HF) with evidence of ischaemic heart disease (IHD); however, there is limited research into cell specific, DNA methylation sensitive genes that are affected by dysregulated DNA methylation patterns. In this study, we aimed to identify DNA methylation sensitive genes in the ischaemic heart and elucidate their role in cardiac fibrosis., Methods: A multi-omics integrative analysis was carried out on RNA sequencing and methylation sequencing on HF with IHD (n = 9) versus non-failing (n = 9) left ventricular tissue, which identified Integrin beta-like 1 (ITGBL1) as a gene of interest. Expression of Itgbl1 was assessed in three animal models of HF; an ischaemia-reperfusion pig model, a myocardial infarction mouse model and an angiotensin-II infused mouse model. Single nuclei RNA sequencing was carried out on heart tissue from angiotensin-II infused mice to establish the expression profile of Itgbl1 across cardiac cell populations. Subsequent in vitro analyses were conducted to elucidate a role for ITGBL1 in human cardiac fibroblasts. DNA pyrosequencing was applied to assess ITGBL1 CpG methylation status in genomic DNA from human cardiac tissue and stimulated cardiac fibroblasts., Results: ITGBL1 was >2-fold up-regulated (FDR adj P = 0.03) and >10-fold hypomethylated (FDR adj P = 0.01) in human HF with IHD left ventricular tissue compared with non-failing controls. Expression of Itgbl1 was up-regulated in three isolated animal models of HF and showed conserved correlation between increased Itgbl1 and diastolic dysfunction. Single nuclei RNA sequencing highlighted that Itgbl1 is primarily expressed in cardiac fibroblasts, while functional studies elucidated a role for ITGBL1 in cardiac fibroblast migration, evident in 50% reduced 24 h fibroblast wound closure occurring subsequent to siRNA-targeted ITGBL1 knockdown. Lastly, evidence provided from DNA pyrosequencing supports the theory that differential expression of ITGBL1 is caused by DNA hypomethylation., Conclusions: ITGBL1 is a gene that is mainly expressed in fibroblasts, plays an important role in cardiac fibroblast migration, and whose expression is significantly increased in the failing heart. The mechanism by which increased ITGBL1 occurs is through DNA hypomethylation., (© 2024 The Author(s). ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFβ-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Stress or excitement is a concern when performing endocrine tests on fractious horses. Sedation may be a solution; however, perturbation of test results may preclude useful information. Thyrotropin-releasing hormone (TRH) is a known stimulator of prolactin, thyroid-stimulating hormone (TSH), melanocyte-stimulating hormone (MSH), and ACTH. Thyrotropin-releasing hormone-induced ACTH is a diagnostic tool for the assessment of endocrinopathies such as pituitary pars intermedia dysfunction. It is unknown if drugs commonly used for sedation alter endocrine responses. The objective of this study was to assess the effects of detomidine (DET) and butorphanol on endocrine responses to TRH. Nine light horse mares were used in a replicated 3 × 3 Latin square with the following treatments: saline, DET, and detomidine + butorphanol (DET/BUT), all administered intravenously at 0.01 mg/kg BW. A 1-wk washout period was allowed between phases, all of which were performed in December. Blood samples were collected at -10 and 0 min before treatment and 5 and 10 min post-treatment. Administration of 1 mg TRH occurred 10 min post-treatment, and blood sampling continued 5, 10, 20, and 30 min post-TRH. Data were analyzed by ANOVA as a replicated Latin square with repeated sampling. Plasma prolactin increased (P < 0.0001) after TRH in all groups, rapidly peaking at 5 min in drug-treated mares and 40 min in saline-treated mares. The peak prolactin response to TRH was 2-fold higher (P < 0.0001) in saline-treated mares compared with those drug-treated. A peak rise in plasma TSH was observed in DET/BUT-treated mares 10 min after TSH and was greater (P ≤ 0.007) compared with DET- and saline-treated mares. Plasma MSH was stimulated (P = 0.001) by DET and DET/BUT before TRH, and the peak MSH response to TRH was greater (P < 0.0001) in drug-treated mares, although not hastened as observed with prolactin and TSH. A peak rise in ACTH was observed in drug-treated mares 5 min after administration of TRH, whereas a peak rise was observed in control mares 10 min post-TRH and was almost 2-fold lower (P = 0.05) than the peak observed in DET and DET/BUT-treated mares. Basal ACTH concentrations were not affected by DET or DET/BUT, indicating that sedation with these compounds may be achieved when needing to measure basal plasma ACTH. Treatment with DET and DET/BUT did alter the prolactin, TSH, MSH, and ACTH responses to TRH; therefore, the use of these drugs may not be advisable when assessing endocrine responses to TRH stimulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, β-epithelial Na + channel-overexpressing transgenic (βENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS -/- ) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of βENaC-Tg mice compared with wild-type (WT) littermates. CatS -/- βENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with βENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of βENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in βENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target., Competing Interests: Conflict of interest: D.M. Small has nothing to disclose. Conflict of interest: R.R. Brown has nothing to disclose. Conflict of interest: D.F. Doherty has nothing to disclose. Conflict of interest: A. Abladey has nothing to disclose. Conflict of interest: Z. Zhou-Suckow has nothing to disclose. Conflict of interest: R.J. Delaney has nothing to disclose. Conflict of interest: L. Kerrigan has nothing to disclose. Conflict of interest: C.M. Dougan has nothing to disclose. Conflict of interest: K.S. Borensztajn has nothing to disclose. Conflict of interest: L. Holsinger is an employee of Virobay. Conflict of interest: R. Booth has a patent US 7,547,701 issued. Conflict of interest: C.J. Scott is a consultant for Fusion Antibodies PLC, outside the submitted work; and is named inventor on various patents on antibodies to cathepsin S for treatment of cancer, many of which have now lapsed as not a current research direction for the company (Fusion Antibodies PLC). Conflict of interest: G. López-Campos has nothing to disclose. Conflict of interest: J.S. Elborn reports personal fees for advisory board work from Bayer, grants and personal fees for advisory board work from Horizion, during the conduct of the study; personal fees for advisory board work from Chiesi and Polyphor, outside the submitted work. Conflict of interest: M.A. Mall reports grants from German Federal Ministry of Education and Research (contract numbers 82DZL00401 and 82DZL004A1), during the conduct of the study; personal fees for advisory board and consultancy work from Spyryx Biosciences, Boehringer Ingelheim and Polyphor, personal fees for advisory board work from ProQR, PTC Pharmaceuticals, Arrowhead and Pro Axis, personal fees for consultancy and lecturing from Bayer, personal fees for consultancy from Enterprise Therapeutics and Sterna Biologicals, personal fees for advisory board work, consultancy and lecturing from Vertex Pharmaceuticals, outside the submitted work; and has a patent on the Scnn1b-transgenic mouse with royalties paid. Conflict of interest: S. Weldon reports grants from Randox and Pfizer UK, outside the submitted work. Conflict of interest: C.C. Taggart reports personal fees for consultancy from Albumedix, and grants from Randox and Pfizer UK, outside the submitted work., (Copyright ©ERS 2019.)

Background: Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells., Methods: Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines., Results: Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells., Conclusions: Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR., (Copyright © 2018 European Cystic Fibrosis Society. All rights reserved.)

Cystic Fibrosis (CF) lung disease is associated with dysregulation of host defence systems, which ultimately disrupts the balance between inflammation and resolution and leaves the host susceptible to repeated infection. However, the mechanisms underlying these defects are complex and continue to garner significant interest among the CF research community. This review explores emerging data on novel aspects of innate host defence with promising biomarker and therapeutic potential for CF lung disease. Improved understanding of inflammation and host defence against pathogens in patients and animal models during the progression of CF lung disease is pivotal for the discovery of new therapeutics that can limit and/or prevent damage from birth., (Copyright © 2017 European Cystic Fibrosis Society. All rights reserved.)

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines., (Copyright © 2012 UICC.)

Authentication of human tissues, cell lines and primary cell cultures (including stem cell preparations) used as therapeutic modalities is often performed using phenotyping and technologies capable of assessing identity to the species level (e.g., isoenzyme analysis and/or karyotyping). This authentication paradigm alone cannot provide assurance that the correct human cell preparation is administered, so careful labeling and tracking of cells from the donor, during manufacture and as part of the final product are also employed. Precise, accurate identification of human cells to the individual donor level could, however, significantly reduce the risks of exposing human subjects to misidentified cells. The availability of a standardized method for achieving this will provide a way to improve the safety profile of human cell-based products by providing assurance that a given lot of cells originated from the intended donor and were not inadvertently mixed or replaced with cells from other donors. In support of this goal, an international team of scientists has prepared a consensus standard on authentication of human cells using short tandem repeat profiling. Associated with the standard itself will be the establishment and maintenance of a public database of short tandem repeat profiles for commonly used cell lines.

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr-activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

Objective: To determine whether retinal ganglion cell death in primary open-angle glaucoma occurs by apoptosis., Methods: Eighteen eyes of 17 subjects with documented primary open-angle glaucoma were compared with 21 control eyes that were group matched for age, race, and sex. Staging of glaucoma severity was performed by histologic optic nerve evaluation. Fixed, paraffin-embedded retinal sections were assayed by the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (UTP)-biotin nick end-labeling) method to detect the internucleosomal DNA fragmentation that is characteristic of apoptosis., Results: A positive TUNEL reaction was observed among ganglion layer cells in 10 of 18 cases with glaucoma, compared with 1 of 11 control cases without confounding systemic disease (5 control eyes were excluded owing to artifactual staining and 4 eyes had confounding systemic disease). Sections containing more than 250,000 cells in the ganglion cell layer were examined in cases and controls. The frequency of TUNEL-positive cells in the ganglion cell layer in cases with glaucoma was 1.76 per 10,000, or 15.2 times greater than the control frequency from individuals without confounding disease (P < .001; 95% CI, 2.46-623). Eyes without glaucoma from subjects with diabetes and amyotrophic lateral sclerosis showed more positive cells than other controls., Conclusion: Apoptosis seems to be a mechanism of cell death in human eyes with primary open-angle glaucoma.

Human mesenchymal stem cells can be isolated from bone marrow aspirates, purified and cultured for many passages without losing their unique properties. One of the hallmarks of stem cells is pluripotency, and human mesenchymal stem cells can be induced to assume phenotypes of mesenchymal tissues including, but not limited to, those of osteocytes, chondrocytes and adipocytes. Due to their ability to form cartilage, bone, fat and other connective tissue, human mesenchymal stem cells have great potential in regenerating diseased or injured tissues. Successful growth of human mesenchymal stem cells is essential to this process, and we have examined the response of human mesenchymal stem cells towards FGF1 and FGF2, two potent growth factors for human tissues. We provide evidence that: 1) human mesenchymal stem cells produce mRNA for receptors for FGF1 and FGF2; 2) these receptors can be detected on the surface of human mesenchymal stem cells; 3) FGF1 and FGF2 increase the rate at which human mesenchymal stem cells proliferate.

Purpose: To compare quantitative and qualitative differences in elastin content in the optic nerve heads of glaucomatous and control human eyes., Methods: Transmission electron microscopy and quantitative histomorphometry on ten control and ten glaucomatous eyes., Results: Elastin fiber complexes in the control lamina cribrosa were smaller and more numerous than in the insertion zone of the sclera immediately surrounding the lamina. Although the density of elastin fibers in the normal lamina was twice that of the insertion zone (P = 0.004), the percent area of the connective tissue matrix occupied by elastin was the same for both zones (P > 0.4). There was no difference between control and glaucomatous eyes in the quantified parameters of elastin content or in the ultrastructure of elastin between control and glaucomatous eyes., Conclusions: The authors demonstrated for the first time that elastin in the normal lamina consists of fibers of smaller diameter than in the adjacent sclera, although the total amount of elastin is similar in both locations. This may provide maximum viscoelasticity within the limited connective tissue beam area of the lamina. Despite using a large number of specimens, the authors again found no differences between normal and glaucomatous eyes in the number or ultrastructural appearance of elastin fibers.

By using dorsal contacts and pinning to quantify play behavior in juvenile rats, it was found that the D2 agonist, quinpirole, reduced both measures of play at doses greater than 0.05 mg/kg. Eticlopride, a D2 antagonist, also reduced both measures of play and blocked the reduction caused by quinpirole. The effect of quinpirole on play was largely unaffected by concurrent administration of either a D1 agonist (SKF 38393) or a D1 antagonist (SCH 23390), suggesting that D1 and D2 receptors are functionally independent with respect to play behavior. Quinpirole also reduced overall activity, suggesting that the effects on play may not be selective to neural circuitry responsible for play behavior. Although low doses of quinpirole (0.001--0.03 mg/kg) had a tendency to increase pinning, this effect was not very robust. These data suggest that D2 dopamine receptors may not have a major role in the control of play behavior in juvenile rats.

Purpose: To investigate whether retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis., Methods: Chronic elevated eye pressure was produced in 20 monkey eyes, and the optic nerve was transected unilaterally in the orbit of 10 monkeys and 14 rabbits. Sixteen monkey and 14 rabbit eyes were studied as normal controls. Analytic methods included light and electron microscopy, histochemistry for DNA fragmentation (TUNEL method), and DNA electrophoresis in agarose gels., Results: Dying ganglion cells in the experimental retinas exhibited morphologic features of apoptosis, including chromatin condensation and formation of apoptotic bodies. Cells with a positive reaction for DNA fragmentation were observed in eyes subjected to axotomy and experimental glaucoma but were only rarely encountered in control eyes. No evidence of internucleosomal fragmentation was detected electrophoretically, possibly because of the small proportion of cells that were dying at any given time., Conclusion: Some retinal ganglion cells injured by glaucoma and by axotomy die by apoptosis.

Purpose: To determine if photoreceptors die in primary open-angle glaucoma., Methods: Retinas were examined in a masked fashion from nine standard locations of 14 eyes with documented open-angle glaucoma and from nine age-matched control eyes. The number and density of photoreceptors, as well as the area and height of the outer nuclear layer, were calculated with an automated image analysis system. The number of photoreceptors per 0.1 mm of retina was determined., Results: No significant difference was seen between control and glaucomatous eyes in comparisons of photoreceptor density, outer nuclear layer height, or photoreceptors per 0.1 mm of retinal length in nine retinal zones. There was no detectable association between photoreceptor number and severity of glaucoma (defined as mild, moderate, or severe), visual field, and optic nerve fiber loss. In eyes in which damage predominated in the upper or lower visual field, no corresponding difference in photoreceptor number in upper compared to lower retinal zones was observed., Conclusion: Photoreceptors are not lost in substantial numbers in primary open-angle glaucoma.

By using a DNase I footprinting assay, we have purified a factor by DNA affinity chromatography that binds to the minimal enhancer region of the Drosophila knirps gene and subsequently identified the protein as the core histone H2B. This inadvertent purification of a core histone as a putative sequence-specific DNA binding protein was due to a previously unknown property of H2B to interact with DNA in a periodic manner. Moreover, we found that each of the individual core histones, but not histone H1 or high mobility group protein 1, bound to the knirps enhancer to give a repetitive DNase I footprint pattern with a periodicity of about 10 base pairs, which is approximately one turn of the DNA helix. In addition, preparations containing the core histones H2A-H2B or H3-H4 yielded identical periodic DNase I footprint patterns on several different promoter and enhancer regions. These findings suggest that there are periodic, homotypic interactions between DNA-bound core histones that result from an alteration of the overall DNA structure such as the curvature rather than a specific sequence. We have also shown that histones H2A-H2B can repress initiation of transcription by RNA polymerase II. The phenomena described here may reflect histone-DNA interactions in non-nucleosomal stretches of chromatin and could be involved in some aspects of either rotational or translational positioning of nucleosomes. Furthermore, these findings indicate that a repeated 10 bp DNase I ladder, which has previously been considered to be a property of an intact nucleosome, can also be generated with subnucleosomal components. It will thus be necessary to reevaluate the criteria applied to the analysis of nucleosomes both in vivo and in vitro.

To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.