Chromatin accessibility profiling by ATAC-seq

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) provides a simple and scalable way to detect the unique chromatin landscape associated with a cell type and how it may be altered by perturbation or disease. ATAC-seq requires a relatively small number of input cells and does not require a priori knowledge of the epigenetic marks or transcription factors governing the dynamics of the system. Here we describe an updated and optimized protocol for ATAC-seq, called Omni-ATAC, that is applicable across a broad range of cell and tissue types. The ATAC-seq workflow has five main steps: sample preparation, transposition, library preparation, sequencing and data analysis. This protocol details the steps to generate and sequence ATAC-seq libraries, with recommendations for sample preparation and downstream bioinformatic analysis. ATAC-seq libraries for roughly 12 samples can be generated in 10 h by someone familiar with basic molecular biology, and downstream sequencing analysis can be implemented using benchmarked pipelines by someone with basic bioinformatics skills and with access to a high-performance computing environment

Molecular Identification of Collagen 17a1 as a Major Genetic Modifier of Laminin Gamma 2 Mutation-Induced Junctional Epidermolysis Bullosa in Mice

<div><p>Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic <i>jeb</i> allele of laminin gamma 2 (<i>Lamc2</i>). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the <i>Lamc2^jeb</i> mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that <i>Col17a1</i> is a strong genetic modifier of the non-Herlitz JEB that develops in <i>Lamc2^jeb</i> mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the <i>Lamc2^jeb</i> mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.</p></div

Reduced congenic mapping limits the modifier to within <i>Col17a1</i>.

<p><b>A</b>, The most informative reduced congenics used in this study. Chr19 donor segments are indicated in black and background strain segments in white. Primer names and chr19 Mb positions are listed above based on C57BL/6J NCBI Build 38. NP, non-polymorphic; NT, not tested. Not to scale. Phenotypes: R, resistant; I, intermediate; S, susceptible. <b>BC</b>, Average ear and tail scores (B) and tail tension measurements at 10 weeks of age (C) for the most informative B6.<i>chr19^AJ</i>, B6.<i>chr19^129S1</i> and B6.<i>chr19^PWD</i> recombinants. <b>D</b>, Ear and tail scores of DBA1.<i>chr19^129X1</i> recombinants. ***, <i>P</i>≤0.001; ****, ≤0.0001, NS, >0.05.</p

Allelic variation limited to chr19 influences the timing of onset of cutaneous phenotypes of JEB.

<p><b>A</b>, Average ear and tail scores, and <b>B</b>, 10 week old tail tension test values comparing B6 mice (most susceptible) with B6.<i>chr19^PWD</i> consomic (most resistant), B6.<i>chr19^A/J</i> consomic, B6.<i>chr19^129S1</i> congenic, and B6 × B6.<i>chr19^B6/PWD</i> heterozygous male mice (intermediate resistance). <b>A</b>, N = 2 to 50 mice per timepoint. *, <i>P</i>≤0.05; ***; ≤0.001; ****, ≤0.0001; NS, >0.05. <b>C</b>, Representative images of ears and tails from 23 week old male B6 and B6.<i>chr19^PWD</i> consomic mice. All mice are <i>Lamc2^jeb/jeb</i>.</p

Nucleotide variation within the 1085<i>Col17a1</i> among phenotyped mouse strains.

<p>Positions of SNPs based on C57BL/6J NCBI Build 38 are indicated. Black, B6 SNP allele; red, other SNP allele;. <b>Bold</b>, uppercase exonic SNPs. Left and right blocks indicate the recombination boundaries of the candidate interval defined by B6/PWD breakpoints: white, B6; black, PWD.</p

The FVB and B6.<i>chr19^PWD</i> backgrounds do not rescue perinatal lethality of <i>Lamc2</i>-null mice.

*<p>Genotyping results from progeny of <i>Lamc2^tm1Uit/+</i> intercross matings of the indicated strain backgrounds.</p>+<p>. Wild-type.</p

Mouse strain background causes substantial variation in the onset and severity of JEB-nH in <i>Lamc2^jeb/jeb</i> mice.

<p><b>A</b>, Average ear and tail scores from 0 (unaffected) to 6 (severe) demonstrate a range of ages of onset. N = 4–50 mice per strain per timepoint. <b>B</b>, Average tail scores for (MRL × FVB) F₁ mice are intermediate compared to the parental strains. N = 5–16 F₁ mice per timepoint. <b>C</b>, Representative ear and tail images of MRL at 8 wks and FVB at 12 and 48 wks demonstrate the typical early onset MRL and late onset FVB phenotypes. <b>D</b>, Representitive fluorescent imaging of cross-section of tail skin of 7–8 week old male mice demonstrates tissue separation (*) at the lamina lucida in MRL which does not occur in age-matched FVB mice. <b>E</b>, Differential tail tension measurements (in Newtons) in mice 6 and 10 weeks of age for the same strains as (A). (datapoints are measurements for individual mice). All mice are male <i>Lamc2^jeb</i>^/j<i>eb</i> homozygotes. *, <i>P</i>≤0.05; ***; ≤0.001; ****, ≤0.0001.</p