Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd

Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy

Chromatin-binding proteins play a crucial part in every aspect of chromatin structure and gene expression. An experimental approach to studying the binding of protein to chromatin in living cells is the use of photobleaching methods. In these experiments a fluorescently tagged protein of interest is introduced into cells and its apparent mobility is measured as an indicator of its dynamic properties. Because nuclear proteins move passively by rapid diffusion through the nuclear space, binding of a protein dramatically affects its overall mobility and therefore the measured mobility contains information about the in vivo binding properties of a protein. Qualitative analysis of photobleaching data gives an impression of whether a protein binds stably or transiently to chromatin in vivo. In addition to the standard qualitative analysis of photobleaching experiments, kinetic modeling methods can be applied for data analysis to permit extraction of quantitative information about simple biophysical properties of chromatin proteins. This method can be used to determine the residence time of a protein on chromatin, the size of the bound and free pools, and the number of kinetically distinct fractions of a protein

The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks

DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks

TBP Dynamics in Living Human Cells: Constitutive Association of TBP with Mitotic Chromosomes

The recruitment of TATA binding protein (TBP) to gene promoters is a critical rate-limiting step in transcriptional regulation for all three eukaryotic RNA polymerases. However, little is known regarding the dynamics of TBP in live mammalian cells. In this report, we examined the distribution and dynamic behavior of green fluorescence protein (GFP)-tagged TBP in live HeLa cells using fluorescence recovery after photobleaching (FRAP) analyses. We observed that GFP-TBP associates with condensed chromosomes throughout mitosis without any FRAP. These results suggest that TBP stably associates with the condensed chromosomes during mitosis. In addition, endogenous TBP and TBP-associated factors (TAFs), specific for RNA polymerase II and III transcription, cofractionated with mitotic chromatin, suggesting that TBP is retained as a TBP-TAF complex on transcriptionally silent chromatin throughout mitosis. In interphase cells, GFP-TBP distributes throughout the nucleoplasm and shows a FRAP that is 100-fold slower than the general transcription factor GFP-TFIIB. This difference supports the idea that TBP and, most likely, TBP-TAF complexes, remain promoter- bound for multiple rounds of transcription. Altogether, our observations demonstrate that there are cell cycle specific characteristics in the dynamic behavior of TBP. We propose a novel model in which the association of TBP-TAF complexes with chromatin during mitosis marks genes for rapid transcriptional activation as cells emerge from mitosis