Genome-wide organization of chromatin and the transcription machinery

Published on February 4, 2014   48 min

Other Talks in the Series: Epigenetics, Chromatin, Transcription and Cancer

0:00
Hello. Today's talk is on the Genome-wide Organization of Chromatin and the Transcriptional Machinery. My name is Dr. Frank Pugh. I'm from the Center for Eukaryotic Gene Regulation at Penn State University in State College, Pennsylvania. As a way of background, I've got my undergraduate degree at Cornell University in Ithaca, New York in 1983 and then went on to graduate school in biochemistry at the University of Wisconsin at Madison, working on genetic recombination. I got my Ph.D. in 1987. I then went on to the University of California at Berkeley where I studied under Dr. Robert Teigen for a postdoc After which in 1991, I started my own lab at Penn State University and have been there ever since. And the focus of my research has been on biochemical and genomic mechanisms of eukaryotic gene regulation.
0:56
Shown here is an image of a typical eukaryotic gene and the proteins that bind to it. The green balls that you see are the nucleosomes which package the chromatin. And you can see by that small black arrow, the TSS, is where the transcriptional start site resides. Between the minus 1 and the plus 1 nucleosomes is an open region where there are no nucleosomes. And that is where the transcription machinery assembles. And there is, perhaps, four stages that you can think of assembly. One is orchestration. That's those red circles that bind to specific DNA sequences at or near the minus 1 nucleosome. The second step involves, perhaps, chromatin remodeling, the rearrangement of proteins on the DNA surface to make the DNA more accessible to other transcription factor binding. So that's step two, access. The third step is the assembly of the general transcriptional machinery in the initiation phase. That's shown in light blue at the promoter nucleosome-free region. And then, that's followed in step four by the recruitment of RNA polymerase in elongation factors that ultimately need to enter into the gene and transcribe the genome. We're going to first talk about the organization of nucleosomes shown here.
2:16
So let's focus in on the nucleosome itself to see how the arrangement of DNA sequences, shown in gold, are organized around the proteinaceous histone core. In this movie, as you see the sequence rotating around, it becomes obvious that there are rotationally-exposed and rotationally-covered sequences of DNA. There's also DNA sequences near the edge of the nucleosome where the DNA exits and enters the nucleosome core particle. One could imagine that as this DNA can rotate on the surface of the histone core, sites become accessible or become occluded. And therefore not available for transcription factor binding. So in order to understand how sequence-specific DNA binding proteins regulate transcription, one needs to know exactly where these nucleosome particles reside in the genome, since movement, little as five base pairs, can alter the rotational phase of the DNA such that a rotationally-exposed site becomes
3:24
So this talk will be broken down into four sections. The first is, how are nucleosome positions determined across a genome? So we'll discuss the methodologies involved in mapping nucleosome positions. Next, we'll discuss what implications nucleosome positions have on transcription initiation mechanisms. Then, we'll briefly look at the function of specific nucleosome positions, both in terms of histone modifications and the factors that might bind to those nucleosomes. And then, I'll finish up with a discussion of a new technique that we developed for ultra-high resolution mapping of where transcription factors bind across the genome and what that tells us about transcription mechanisms.
4:14
The human genome has approximately 10 million nucleosomes. Where as the yeast genome has only about 60,000. The question is, where are those nucleosome located? And the way we map them is as follows. First, we trap the nucleosome in their in vivo position, using formaldehyde that cross-links protein to DNA. The second step is, then, we isolate chromatin. And we digest them with MNase, micrococcal nuclease. Micrococcal nuclease digests the linker DNA, DNA between nucleosome leaving the DNA on the nucleosome largely protected. And so that creates, perhaps, a protected fragment of DNA that we're ultimately going to sequence. But first, we want to make sure that we've got a nucleosome. So we immunopurify a nucleosome using antibodies against histone H3, that's Chromatin immunoprecipitation. And then finally, we sequence the DNA using deep sequencing technology, illumina sequencer, or applied biosystem solid sequencing, where you basically sequence the ends of the DNA shown by the blue and red arrows. Now, it's the five prime end or the first nucleotide of that read that defines the nucleosome border.
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Genome-wide organization of chromatin and the transcription machinery

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