Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization

Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.Eukaryoten nutzen Nukleosomen um das Packvolumen ihres Genoms drastisch zu reduzieren. Dadurch wird die Zugänglichkeit der DNA stark eingeschränkt. Um bei Bedarf DNA-Abschnitte von Nukleosomen zu lösen nutzen eukaryotische Zellen ATP-abhängige Remodeling Faktoren. Zwei dieser Remodeling Faktoren, ACF und CHRAC, sind Multiproteinkomplexe und beinhalten als grösste Untereinheit das Protein ACF1. Beide Komplexe können Nukleosomen assemblieren, verschieben und mit gleichmässigen Abständen auf einem DNA-Strang anordnen. Es ist aber noch weitgehend unbekannt, was die genauen Funktionen dieser Komplexe in der lebenden Zelle sind, und wie sie dort die Nukleosomenorganisation beeinflussen. Um diese Fragen zu beantworten habe ich Nukleosomenverteilungen und Transkriptionsprofile in normalen und ACF1-mutanten Drosophilaembryonen bestimmt. Zusätzlich habe ich mit Hilfe der Chromatin-Immunopräzipitations-Methode (ChIP) untersucht, wo auf dem Genome ACF1 bindet. Die Nuklesomenverteilung wurde bestimmt, indem Embryonenchromatin mit MNase verdaut wurde, und die resultierenden DNA-Fragmente sequenziert wurden. Dabei haben wir herausgefunden, dass es verschiedene Regionen auf dem Genom mit sehr gleichmässig angeordneten Nukleosomen gibt. Wir nennen diese Regionen “regularity regions”. Sie sind sowohl in transkribiertem wie auch inaktivem Chromatin zu finden und haben unterschiedliche Nukleosomenlinkerlängen. Viele dieser regulären Regionen sind nur in normalen, aber nicht in ACF1-mutanten Embryonen zu finden. Diese ACF1-abhängigen regulären Chromatinabschnitte zeigen auch bislang unbekanntes DNA Motiv. Indem die Nukleosomenperiodizität mit einer Autokorrelationsfunktion bestimmt wurde, konnte gezeigt werden, dass der Nukleosomenlinker in ACF1-mutanten Embryonen länger als normal ist. Obwohl klare Defekte in der Chromatinorganisation in ACF1 Mutanten gefunden wurden, zeigte eine RNA-Expressionsanalyse keine grösseren Unterschiede zu normalen Embryonen. Die genomweite Verteilung von ACF1 wurde mit der ChIP-Methode untersucht. Wir haben dabei eine starke Anreicherung von ACF1 an aktiven Promotoren gefunden, sehr ähnlich wie beim verwandten Remodeling Faktor RSF-1. Nachdem wir aber die Experimente in ACF1 und RSF-1 mutanten Embryonen wiederholt hatten, und die Lokalisierung beider Faktoren an Promotoren immer noch zu sehen war, schlossen wir daraus, dass die beobachteten Anreicherungen falsch-positiv waren. Insgesamt haben wir in unseren ChIP-Experimenten an 3100 Stellen des Genoms falsch-positive “Phantom Peaks” gefunden. Indem wir verschiedene öffentlich zugängliche ChIP-Profile mit diesen “Phantom Peaks” verglichen haben, konnten wir zeigen, dass mehrere dieser Profile ein grosse Übereinstimmung mit den “Phantom Peaks” hatten. Wir haben also einerseits innerhalb des Fliegengenoms Regionen mit sehr gleichmässig angeordneten Nukleosomen gefunden und gezeigt, dass ein Teil davon die Regularität in der Absenz von ACF1 verliert. Darüberhinaus haben wir eine Klasse von falsch-positiven ChIP-Anreicherungen an aktiven Promotoren identifiziert: Diese Liste von “Phantom Peaks” kann nun dazu benutzt werden, potentiell falsch-positive Signale in ChIP-Experimenten zu identifizieren, vor allem wenn keine Kontrolle mit einer Nullmutante durchgeführt werden kann

Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization

Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.Eukaryoten nutzen Nukleosomen um das Packvolumen ihres Genoms drastisch zu reduzieren. Dadurch wird die Zugänglichkeit der DNA stark eingeschränkt. Um bei Bedarf DNA-Abschnitte von Nukleosomen zu lösen nutzen eukaryotische Zellen ATP-abhängige Remodeling Faktoren. Zwei dieser Remodeling Faktoren, ACF und CHRAC, sind Multiproteinkomplexe und beinhalten als grösste Untereinheit das Protein ACF1. Beide Komplexe können Nukleosomen assemblieren, verschieben und mit gleichmässigen Abständen auf einem DNA-Strang anordnen. Es ist aber noch weitgehend unbekannt, was die genauen Funktionen dieser Komplexe in der lebenden Zelle sind, und wie sie dort die Nukleosomenorganisation beeinflussen. Um diese Fragen zu beantworten habe ich Nukleosomenverteilungen und Transkriptionsprofile in normalen und ACF1-mutanten Drosophilaembryonen bestimmt. Zusätzlich habe ich mit Hilfe der Chromatin-Immunopräzipitations-Methode (ChIP) untersucht, wo auf dem Genome ACF1 bindet. Die Nuklesomenverteilung wurde bestimmt, indem Embryonenchromatin mit MNase verdaut wurde, und die resultierenden DNA-Fragmente sequenziert wurden. Dabei haben wir herausgefunden, dass es verschiedene Regionen auf dem Genom mit sehr gleichmässig angeordneten Nukleosomen gibt. Wir nennen diese Regionen “regularity regions”. Sie sind sowohl in transkribiertem wie auch inaktivem Chromatin zu finden und haben unterschiedliche Nukleosomenlinkerlängen. Viele dieser regulären Regionen sind nur in normalen, aber nicht in ACF1-mutanten Embryonen zu finden. Diese ACF1-abhängigen regulären Chromatinabschnitte zeigen auch bislang unbekanntes DNA Motiv. Indem die Nukleosomenperiodizität mit einer Autokorrelationsfunktion bestimmt wurde, konnte gezeigt werden, dass der Nukleosomenlinker in ACF1-mutanten Embryonen länger als normal ist. Obwohl klare Defekte in der Chromatinorganisation in ACF1 Mutanten gefunden wurden, zeigte eine RNA-Expressionsanalyse keine grösseren Unterschiede zu normalen Embryonen. Die genomweite Verteilung von ACF1 wurde mit der ChIP-Methode untersucht. Wir haben dabei eine starke Anreicherung von ACF1 an aktiven Promotoren gefunden, sehr ähnlich wie beim verwandten Remodeling Faktor RSF-1. Nachdem wir aber die Experimente in ACF1 und RSF-1 mutanten Embryonen wiederholt hatten, und die Lokalisierung beider Faktoren an Promotoren immer noch zu sehen war, schlossen wir daraus, dass die beobachteten Anreicherungen falsch-positiv waren. Insgesamt haben wir in unseren ChIP-Experimenten an 3100 Stellen des Genoms falsch-positive “Phantom Peaks” gefunden. Indem wir verschiedene öffentlich zugängliche ChIP-Profile mit diesen “Phantom Peaks” verglichen haben, konnten wir zeigen, dass mehrere dieser Profile ein grosse Übereinstimmung mit den “Phantom Peaks” hatten. Wir haben also einerseits innerhalb des Fliegengenoms Regionen mit sehr gleichmässig angeordneten Nukleosomen gefunden und gezeigt, dass ein Teil davon die Regularität in der Absenz von ACF1 verliert. Darüberhinaus haben wir eine Klasse von falsch-positiven ChIP-Anreicherungen an aktiven Promotoren identifiziert: Diese Liste von “Phantom Peaks” kann nun dazu benutzt werden, potentiell falsch-positive Signale in ChIP-Experimenten zu identifizieren, vor allem wenn keine Kontrolle mit einer Nullmutante durchgeführt werden kann

Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as 'Phantom Peaks'. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted

Modulation of morphology and efficacy of new CB1 receptor antagonist using simple and benign polymeric additives

1014-1021The compound 1, [(1H-[1]benzoxepino[5,4-c]pyrazole-3-carboxamide, 8-chloro-1-(2,4-dichlorophenyl)-4,5-dihydro-N- 1-piperidinyl], a known CB1 modulator has been synthesized and characterized by IR, NMR and single Crystal X-ray study. The single crystal study of 1 displays a number of halogen bonds leading to 1-D network along with other weak noncovalent interactions. The CB1 modulator 1 inherently possesses extremely low solubility in water, which makes its application as drug difficult, and this may be attributed to multiple halogen bonds present in the crystal structure. A series of polymer additives, which are Generally Regarded As Safe (GRAS), have been explored to investigate whether they can modulate the halogen bond present in 1 through formation of various non-bonded interactions. Surprisingly, these polymers are found to change crystal morphology, crystal packing while retaining efficacy and bioavailability. The polymer molecular weight is found to play a significant role in crystal morphology modification especially in case of polyethylene glycol (PEG). The formation of new polymorphic forms of 1 and modification of halogen bond has been established using powder X-ray diffraction and IR study, respectively, in case of PEG 4000, PVPK-30, PVA polymers and compound 1 adducts

Modulation of morphology and efficacy of new CB1 receptor antagonist using simple and benign polymeric additives

The compound 1, [(1H-[1]benzoxepino[5,4-c]pyrazole-3-carboxamide, 8-chloro-1-(2,4-dichlorophenyl)-4,5-dihydro-N-1-piperidinyl], a known CB1 modulator has been synthesized and characterized by IR, NMR and single Crystal X-ray study. The single crystal study of 1 displays a number of halogen bonds leading to 1-D network along with other weak non-covalent interactions. The CB1 modulator 1 inherently possesses extremely low solubility in water, which makes its application as drug difficult, and this may be attributed to multiple halogen bonds present in the crystal structure. A series of polymer additives, which are Generally Regarded As Safe (GRAS), have been explored to investigate whether they can modulate the halogen bond present in 1 through formation of various non-bonded interactions. Surprisingly, these polymers are found to change crystal morphology, crystal packing while retaining efficacy and bioavailability. The polymer molecular weight is found to play a significant role in crystal morphology modification especially in case of polyethylene glycol (PEG). The formation of new polymorphic forms of 1 and modification of halogen bond has been established using powder X-ray diffraction and IR study, respectively, in case of PEG 4000, PVPK-30, PVA polymers and compound 1 adducts.

Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing.

Funder: Ludwig Center at HarvardFunder: National Cancer Institute: K22CA193848Funder: US National Institutes of Health Intramural Research Program Project Z1AES103266Chromothripsis is a mutational phenomenon characterized by massive, clustered genomic rearrangements that occurs in cancer and other diseases. Recent studies in selected cancer types have suggested that chromothripsis may be more common than initially inferred from low-resolution copy-number data. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we analyze patterns of chromothripsis across 2,658 tumors from 38 cancer types using whole-genome sequencing data. We find that chromothripsis events are pervasive across cancers, with a frequency of more than 50% in several cancer types. Whereas canonical chromothripsis profiles display oscillations between two copy-number states, a considerable fraction of events involve multiple chromosomes and additional structural alterations. In addition to non-homologous end joining, we detect signatures of replication-associated processes and templated insertions. Chromothripsis contributes to oncogene amplification and to inactivation of genes such as mismatch-repair-related genes. These findings show that chromothripsis is a major process that drives genome evolution in human cancer

Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing

Funder: Ludwig Center at HarvardFunder: National Cancer Institute: K22CA193848Funder: US National Institutes of Health Intramural Research Program Project Z1AES103266Abstract: Chromothripsis is a mutational phenomenon characterized by massive, clustered genomic rearrangements that occurs in cancer and other diseases. Recent studies in selected cancer types have suggested that chromothripsis may be more common than initially inferred from low-resolution copy-number data. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we analyze patterns of chromothripsis across 2,658 tumors from 38 cancer types using whole-genome sequencing data. We find that chromothripsis events are pervasive across cancers, with a frequency of more than 50% in several cancer types. Whereas canonical chromothripsis profiles display oscillations between two copy-number states, a considerable fraction of events involve multiple chromosomes and additional structural alterations. In addition to non-homologous end joining, we detect signatures of replication-associated processes and templated insertions. Chromothripsis contributes to oncogene amplification and to inactivation of genes such as mismatch-repair-related genes. These findings show that chromothripsis is a major process that drives genome evolution in human cancer

Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as 'Phantom Peaks'. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted

Utility of cerebral oximetry in balloon mitral valvotomy and its correlation with post-procedure neurological complications: A pragmatic prospective observational study

Background and Aims: Neurological complications (NCs) are significantly associated with reduced regional cerebral saturation (rSO2) in patients undergoing cardiac surgeries, as assessed with cerebral oximetry (COx). However, limited evidence is available in patients undergoing balloon mitral valvotomy (BMV). Thus, we evaluated the utility of COx in patients undergoing BMV, the incidence of BMV-related NCs and the association of >20% reduction in rSO2 with NCs. Methods: This pragmatic, prospective, observational study was performed after ethical approval, over November 2018 to August 2020, in the cardiology catherization laboratory of a tertiary care hospital. The study involved 100 adult patients undergoing BMV for symptomatic mitral stenosis. The patients were evaluated at initial presentation, pre-BMV, post-BMV and 3 months after the BMV. Results: The incidence of NCs was 7%, including transient ischaemic attack (n = 3), slurred speech (n = 2) and hemiparesis (n = 2). A significantly greater proportion of patients with NCs had a > 20% decrease in the rSO2 (P value = 0.020). At >20% cut-off, the COx had a sensitivity and specificity of 57.1% and 80%, respectively, in the prediction of NCs. Female sex (P value = 0.039), history of cerebrovascular episodes (P value < 0.001) and number of balloon attempts (P value < 0.001) were significantly associated with NCs. Patients with and without NCs had a significantly greater post-BMV mean % change in rSO2 than pre-BMV (both right and left sides), but the magnitude of mean % change was greater in those with NCs. Conclusions: COx alone has low sensitivity and specificity in the prediction of NCs and cannot reliably predict the development of post-BMV NCs