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ABSTRACT: Chromatin immunoprecipitation (ChIP) followed by deep sequencing can now easily be performed across different conditions, time points and even species. However, analyzing such data is not trivial and standard methods are as yet unavailable. Here we present a protocol to systematically compare ChIP-sequencing (ChIP-seq) data across conditions. We first describe technical guidelines for data preprocessing, read mapping, read-density visualization and peak calling. We then describe methods and provide code with specific examples to compare different data sets across species and across conditions, including a threshold-free approach to measure global similarity, a strategy to assess the binary conservation of binding events and measurements for quantitative changes of binding. We discuss how differences in binding can be related to gene functions, gene expression and sequence changes. Once established, this protocol should take about 2 d to complete and be generally applicable to many data sets.
Nature Protocol 01/2012; 7(1):45-61. · 8.36 Impact Factor
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ABSTRACT: The binding of some transcription factors has been shown to diverge substantially between closely related species. Here we show that the binding of the developmental transcription factor Twist is highly conserved across six Drosophila species, revealing strong functional constraints at its enhancers. Conserved binding correlates with sequence motifs for Twist and its partners, permitting the de novo discovery of their combinatorial binding. It also includes over 10,000 low-occupancy sites near the detection limit, which tend to mark enhancers of later developmental stages. These results suggest that developmental enhancers can be highly evolutionarily constrained, presumably because of their complex combinatorial nature.
Nature Genetics 05/2011; 43(5):414-20. · 35.53 Impact Factor
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ABSTRACT: The packaging of eukaryotic DNA into chromatin has profound consequences for gene regulation, as well as for other DNA transactions such as recombination, replication and repair. Understanding how this packaging is determined is consequently a pressing problem in molecular genetics. DNA sequence, chromatin remodelers and transcription factors affect chromatin structure, but the scope of these influences on genome-wide nucleosome occupancy patterns remains uncertain. Here, we use high resolution tiling arrays to examine the contributions of two general regulatory factors, Abf1 and Rap1, to nucleosome occupancy in Saccharomyces cerevisiae. These factors have each been shown to bind to a few hundred promoters, but we find here that thousands of loci show localized regions of altered nucleosome occupancy within 1 h of loss of Abf1 or Rap1 binding, and that altered chromatin structure can occur via binding sites having a wide range of affinities. These results indicate that DNA-binding transcription factors affect chromatin structure, and probably dynamics, throughout the genome to a much greater extent than previously appreciated.
Nucleic Acids Research 11/2010; 39(6):2032-44. · 8.03 Impact Factor
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ABSTRACT: Mediator is a large, multisubunit complex that is essential for transcription of mRNA by RNA Pol II in eukaryotes and is believed to bridge transcriptional activators and the general transcription machinery. However, several recent studies suggest that the requirement for Mediator during transcriptional activation is not universal, but rather activator dependent, and may be indirect for some genes. Here we have investigated Mediator association with several constitutively transcribed genes in yeast by comparing a yeast strain that harbors a temperature-sensitive mutation in an essential Mediator subunit, Srb4, with its wild-type (WT) counterpart. We find modest association of Mediator with constitutively active genes and show that this association is strongly decreased in srb4 ts yeast, whereas association with a nontranscribed region or repressed gene promoters is lower and unaffected in the mutant yeast. The tail module of Mediator remains associated with ribosomal protein (RP) gene promoters in srb4 ts yeast, while subunits from the head and middle modules are lost. Tail module association at Rap1-dependent gene promoters is lost in rap1 ts yeast, indicating that Rap1 is required for Mediator recruitment at these gene promoters and that its recruitment occurs via the tail module. Pol II association is also rapidly and severely affected in srb4 ts yeast, indicating that Mediator is directly required for pol II association at constitutively transcribed genes. Our results are consistent with Mediator functioning as a general transcription factor in yeast.
Proceedings of the National Academy of Sciences 09/2009; 106(39):16734-9. · 9.68 Impact Factor
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ABSTRACT: The histone H3 amino terminus, but not that of H4, is required to prevent the constitutively bound activator Cha4 from remodeling chromatin and activating transcription at the CHA1 gene in Saccharomyces cerevisiae. Here we show that neither the modifiable lysine residues nor any specific region of the H3 tail is required for repression of CHA1. We then screened for histone H3 mutations that cause derepression of the uninduced CHA1 promoter and identified six mutants, three of which are also temperature-sensitive mutants and four of which exhibit a sin(-) phenotype. Histone mutant levels were similar to that of wild-type H3, and the mutations did not cause gross alterations in nucleosome structure. One specific and strongly derepressing mutation, H3 A111G, was examined in depth and found to cause a constitutively active chromatin configuration at the uninduced CHA1 promoter as well as at the ADH2 promoter. Transcriptional derepression and altered chromatin structure of the CHA1 promoter depend on the activator Cha4. These results indicate that modest perturbations in distinct regions of the nucleosome can substantially affect the repressive function of chromatin, allowing activation in the absence of a normal inducing signal (at CHA1) or of Swi/Snf (resulting in a sin(-) phenotype).
Eukaryotic Cell 10/2008; 7(10):1649-60. · 3.60 Impact Factor
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ABSTRACT: Mediator complex is essential for transcription by RNA polymerase II in eukaryotes. Although chromatin remodeling is an integral part of transcriptional activation at many promoters, whether Mediator is required for this function has not been determined. Here we have used the yeast CHA1 gene to study the role of Mediator in chromatin remodeling and recruitment of the transcription machinery. We show by chromatin immunoprecipitation that Mediator subunits are recruited to the induced CHA1 promoter. Inactivation of Mediator at 37 degrees C in yeast harboring the srb4-138 (med17) ts mutation severely reduces CHA1 activation and prevents recruitment to the induced CHA1 promoter of Med18/Srb5, from the head module of Mediator, and Med14/Rgr1, which bridges the middle and tail modules. In contrast, recruitment of Med15/Gal11 from the tail module is unaffected in med17 ts yeast at 37 degrees C. Recruitment of TATA-binding protein (TBP) is severely compromised in the absence of functional Mediator, whereas Kin28 and polymerase II recruitment are reduced but to a lesser extent. Induced levels of histone H3K4me3 at the CHA1 promoter are not diminished by inactivation of Mediator, whereas recruitment of Paf1 and of Ser2- and Ser5-phosphorylated forms of Rbp1 are reduced but not eliminated. Loss of histone H3 from the induced CHA1 promoter is seen in wild type yeast but is greatly reduced by loss of intact Mediator. In contrast, Swi/Snf recruitment and nucleosome remodeling are unaffected by loss of Mediator function. Thus, Mediator is required for recruitment of the transcription machinery subsequent to chromatin remodeling during CHA1 induction.
Journal of Biological Chemistry 03/2008; 283(9):5276-86. · 4.77 Impact Factor
