The chromatin immunoprecipitation (ChIP) assay is a major tool in the study of genomic processes in vivo. This and other methods are revealing that control of gene expression, cell division and DNA repair involves multiple proteins and great number of their modifications. ChIP assay is traditionally done in test tubes limiting the ability to study signaling of the complex genomic events. To increase the throughput and to simplify the assay we have developed a microplate-based ChIP (Matrix ChIP) method, where all steps from immunoprecipitation to DNA purification are done in microplate wells without sample transfers. This platform has several important advantages over the tube-based assay including very simple sample handling, high throughput, improved sensitivity and reproducibility, and potential for automation. 96 ChIP measurements including PCR can be done by one researcher in one day. We illustrate the power of Matrix ChIP by parallel profiling 80 different chromatin and transcription time-course events along an inducible gene including transient recruitment of kinases.
BackgroundAs a consequence of acute kidney injury (AKI), proximal tubular cells hyperrespond to endotoxin (lipopolysaccharide, LPS) by exaggerated renal Tnf-α Production. This LPS hyperresponsiveness is transcriptionally mediated. The epigenetic pathways that control these responses are unknown.Methods/FindingsWe applied multiplex chromatin immunoprecipitation platform (Matrix ChIP) to explore epigenetic pathways that underlie endotoxin hyperresponsiveness in the setting of preceding unilateral renal ischemia/reperfusion (I/R) in mouse AKI model. Endotoxin exposure after I/R resulted in enhanced transcription, manifested by hyperresponsive recruitment of RNA polymerase II (Pol II) at the Tnf-α gene. At this locus, LPS but not I/R increased levels of Pol II C-terminal domain (CTD) phosho-serine2 &5 and induced dephosphorylation of the transcription-repressive histone H4 phospho-serine-1. In contrast, I/R but not LPS increased the transcription-permissive histone phosphorylation (H3 phospho-serine-10, H3.3 phospho-serine-31) at the Tnf-α gene. In agreement with these observations, I/R but not LPS increased activity of cognate kinases (Erk1/2, Msk1/2 and Aurora A) at the Tnf-α locus. Cross-talk of histone phosphorylation and acetylation synergize to active gene expression. I/R and LPS increased histone acetylation. (H3K9/14Ac, H4K5/8/12/16Ac, H2KA5Ac, H2BK4/7Ac). Levels of some histone acetyltransferases at this gene (PCAF and MOF) were increased by I/R but not by LPS, while others were induced by either I/R or LPS and exhibited endotoxin hyperresponsive patterns (GCN5, CBP and p300). The adaptor protein 14-3-3 couples histone phosphorylation with acetylation, and tethers chromatin modifiers/transcription elongation factors to target genes. Both I/R and LPS increased levels of 14-3-3 and several chromatin/transcription modifiers (BRD4, BRG1, HP-1γ and IKKα) at the Tnf-α gene, all exhibiting endotoxin hyperresponsive recruitment patterns similar to Pol II.ConclusionsOur results suggest that I/R and LPS differentially trigger phosphorylation (Pol II and histone) and acetylation (histone) epigenetic pathways that interact at the Tnf-α gene to generate endotoxin hyperresponse in AKI.
The laminin ␥1 chain, a critical component of the extracellular matrix, is encoded by the 125-kb-long Lamc1 locus. We profiled RNA polymerase II (Pol II) and histone modifications along the Lamc1 locus to explore transcription of this gene in its native chromatin environment. Treatment with 12-O-tetradecanoylphorbol-13-acetate increased Lamc1 mRNA in rat mesangial cells (RMC). This increase was matched by an increase in Pol II density along the entire length of the Lamc1 locus. In contrast, in the hepatocarcinoma cell line (HTC-IR) an increase in Pol II density was restricted to the promoter and was not followed by mRNA induction. The pattern of histone H3 methylation was similar for both cell types but an increase in H3 lysine 9 acetylation observed at the 5Ј-end was weaker in HTC-IR cells than in RMC. All of the histone modifications showed spatial patterns where levels differed greatly between the 5Ј-and 3Ј-ends of Lamc1. Conversely, at the short, highly induced egr-1 gene the differences in chromatin marks between the 5Ј-and 3Ј-ends were much smaller. The results of this study suggest that 1) Lamc1 transcription can be controlled after transcription initiation, to our knowledge, the first time this has been shown in an extracellular matrix gene, and 2) the length of a gene is a factor that can affect the chromatin environment for Pol II elongation. Fast ChIP; extracellular matrix; hnRNP K; histone; elongation; egr-1 GENE TRANSCRIPTION is composed of a sequence of complex events, which have been conceptually viewed as three discreet phases: initiation, elongation, and termination. Transcription initiation is the most studied phase, and it consists of highly regulated steps that result in the assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II) into a preinitiation complex (PIC) at the core promoter (63). Transcription elongation is a process that follows initiation by which Pol II moves through the gene and synthesizes RNA (57). The termination step consists of cleavage of nascent mRNA, polyadenylation of the mRNA 3Ј-end, and its release from the DNA template.Laminins are major basement membrane proteins that form heterotrimers composed of ␣, , and ␥ chains (3). In the extracellular matrix (ECM), the ␥1 chain, which is found in 10 of the 16 known trimeric laminin isoforms, is the most widely expressed laminin chain (3, 64). Laminin ␥1 is required for endodermal differentiation and its absence causes early lethality in mouse embryos (40,59). Also, expression of laminin ␥1 is increased in cultured mesangial cells exposed to high concentrations of glucose (47, 50) and is one component of the increased ECM expression seen in models of diabetic glomerulosclerosis (15). These and other observations suggest that the molecular composition of laminins is critical (2, 36), that misexpression of laminin ␥1 may be a factor in renal disease, and that laminin ␥1 is an essential component of the ECM which orchestrates developmental events (40, 59).Because of their importance, expression of ␥1 and...
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