Figure 1: Distinct chromatin structures at the promoters of Cga and Lhb appear responsible for their expression patterns.
From: H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes
(a–c) Native chromatin in (a) LβT2 cells that express both Cga and Lhb, and (b) MEFs that do not express them was digested with MNase and subjected to qPCR (MNase-qPCR) with primers amplifying ∼100 bp non-overlapping fragments spanning ∼−550/+250 bp of each gene. (c) For high-coverage nucleosome maps in LβT2 cells, MNase-digested LβT2 DNA was subjected to qPCR with primers amplifying ∼70 bp overlapping fragments. MNase protection (that is, amount of DNA amplified in qPCR reaction after MNase digestion of genomic DNA for each primer pair) is plotted for each amplicon centre relative to genomic coordinates of each gene respective to its TSS. (d) ChIP for Pol2 was carried out in LβT2 cells, using the same sets of primers as in a and b. The levels of precipitated DNA are presented relative to the levels in input samples. (e) Total RNA from LβT2 cells was reverse transcribed with random primers before qPCR. RNA levels were quantitated using a standard curve of plasmid Cga and Lhb cDNA, normalized to RPL0P mRNA and are presented relative to the levels of Lhb. Data shown as mean±s.e.m., n=3–5; P=1.3 × 10−4, two-sample Student’s t-test. ***P<0.001. (f) LβT2 cells were transfected with Cga (−507/+46) or Lhb (−755/+6) DNA sequences fused to the luciferase reporter gene. The levels of firefly luciferase were normalized to those of Renilla luciferase and are expressed as fold over Lhb-driven luciferase activity. Data shown as mean±s.e.m., n=4; P=0.03, two-sample Student’s t-test. *P<0.05.
