A practical guide for DNase-seq data analysis: from data management to common applications

Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon. However, exploring DNase-seq data requires sophisticated downstream bioinformatics analyses. In this study, we first review computational methods for all of the major steps in DNase-seq data analysis, including experimental design, quality control, read alignment, peak calling, annotation of cis-regulatory elements, genomic footprinting and visualization. The challenges associated with each step are highlighted. Next, we provide a practical guideline and a computational pipeline for DNase-seq data analysis by integrating some of these tools. We also discuss the competing techniques and the potential applications of this pipeline for the analysis of analogous experimental data. Finally, we discuss the integration of DNase-seq with other functional genomics techniques.

Distinct Chromatin Configurations Regulate the Initiation and the Maintenance of hGH Gene Expression

For many mammalian genes, initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone ( hGH-N ) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories, histone acetylation at the hGH-N promoter, and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

In Vitro Reconstitution of PHO5 Promoter Chromatin Remodeling Points to a Role for Activator-Nucleosome Competition In Vivo

ABSTRACT The yeast PHO5 promoter is a classical model for studying the role of chromatin in gene regulation. To enable biochemical dissection of the mechanism leading to PHO5 activation, we reconstituted the process in vitro. Positioned nucleosomes corresponding to the repressed PHO5 promoter state were assembled using a yeast extract-based in vitro system. Addition of the transactivator Pho4 yielded an extensive DNase I-hypersensitive site resembling induced PHO5 promoter chromatin. Importantly, this remodeling was energy dependent. In contrast, little or no chromatin remodeling was detected at the PHO8 or PHO84 promoter in this in vitro system. Only the PHO5 promoter harbors a high-affinity intranucleosomal Pho4 binding site (UASp) where Pho4 binding can compete with nucleosome formation, prompting us to test the importance of such competition for chromatin remodeling by analysis of UASp mutants in vivo. Indeed, the intranucleosomal location of the UASp element was critical, but not essential, for complete remodeling at the PHO5 promoter in vivo. Further, binding of just the Gal4 DNA binding domain to an intranucleosomal site could increase PHO5 promoter opening. These data establish an auxiliary role for DNA binding competition between Pho4 and histones in PHO5 promoter chromatin remodeling in vivo.