Bone marrow-derived macrophages (BMDMs)
Macrophages can be obtained from mouse bone marrow by in vitro differentiation of hematopoietic progenitors in the presence of M-CSF. Mature BMDMs respond to various bacterial and viral stimuli, which allows us to analyze inducible gene expression by stimulating cells growing in culture with LPS. In addition, we can limit certain factors, such as pioneer transcription factors and nucleosome remodelers, in differentiating cells and determine the effect this has on nucleosome occupancy and gene induction in mature macrophages. The lab also uses other primary cells (e.g., splenic B-cells, fibroblasts) as well as cell-lines to obtain insight into cell-type specific gene regulation.
The quantitative nucleosome occupancy assay
We are using a quantitative assay that was developed by (Bryant et al. 2008) in yeast to determine fractional nucleosome occupancies at genomic locations of interest in a population of cells. The assay uses sensitivity of cross-linked chromatin to increasing concentrations of MNase to quantify nucleosome occupancy. MNase digestion data is quantified by qRT-PCR followed by fitting of the data to two-state exponential curves
d[DNA]/d[MN] = (1-fr2)e-(k1MN) + fr2e-(k2MN) where k1 is the rate of digestion of naked DNA, k2 is the rate of digestion of nucleosomal DNA and fr2 is the fraction of DNA that is nucleosomal. Curve-fitting is performed using a MATlab protocol. To determine nucleosome occupancy in a region of interest this analysis is performed with overlapping primer pairs and occupancy data is displayed in a Genome Browser (i.e., IGB or IGV) to visualize nucleosome binding.
The Floer lab uses a semi high-throughput pipeline to quantify DNA for nucleosome occupancy measurements. A Tecan Freedom EVO100 robotic liquid handler prepares 384-well qRT-PCR plates and delivers them to a Roche Lightcycler 480. The use of our own SYBR Green chemistry for qRT-PCR analysis allows us to determine nucleosome occupancy at many different locations at a reasonable cost. qRT-PCR data is processed using the Roche Lightcycler 480 software, and is then imported into a MATlab protocol for further analysis. This set-up is also used for other applications including ChIP experiments and mRNA analysis.
Knockdown of target genes by lentiviral delivery of shRNAs in hematopoietic progenitors
A lentiviral shRNA delivery approach allows us to knockdown nucleosome remodelers and transcription factors in hematopoietic progenitors and to determine the effect this has on nucleosome occupancy and gene expression in mature macrophages. A main focus in the lab is to understand the role of the Swi/Snf remodelers BAF and PBAF in controlling nucleosome occupancy at pro-inflammatory gene enhancers. Our hypothesis is that nucleosome remodelers are recruited to macrophage-specific genes by lineage-specific transcription factors during macrophage differentiation, which allows induction of these genes in mature cells.