Speaker: Francesca Mattiroli
Location: TU Delft
Date: 10 February 2017
Author: Carolien Bastiaanssen
To fit the enormous amount of DNA in the restricted volume of the nucleus, the DNA is compacted (Figure 1). This compacted form of DNA is known as chromatin and to enable the organization of DNA into chromatin, the DNA is wrapped around nucleosomes. Each nucleosomes consists of eight histones: an (H3-H4)2 tetramer and two H2A-H2B dimers (Figure 2). All histones have N-terminal tails that protrude out of the nucleosome and form targets for posttranslational modifications. These histone modifications, but also histone variants, can have an inhibiting or promoting effect on DNA interacting proteins. Furthermore, the nucleosomes act as a physical barrier, thereby regulating genome accessibility. During replication, the DNA has to be made accessible thus the nucleosomes are disassembled in front of the replisome and reassembled behind it. Francesca Mattiroli is interested in what the evolutionary origin is of the nucleosome and how eukaryotic chromatin is propagated during DNA replication.
Figure 1: The DNA double helix is wrapped around nucleosomes, that consist of histones. The nucleosomes are further organized to form chromatin. Source: Baylin, S. B. et al. (2007), Genomic biology: The epigenomic era opens, Nature 448, 548-549
Figure 2: Structure of a nucleosome. The DNA is wrapped around a histone octamer which consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, with the histone tails protruding outwards. Adapted from: Petesch, S.J. et al. (2012), Overcoming the nucleosome barrier during transcript elongation, Trends in Genetics, Vol. 28, No.6
The first part of the talk covered the evolution of eukaryotic chromatin structure. Mattiroli and her colleagues found the crystal structure of archaeal chromatin. They found that Archaea possess histone dimers that are similar to eukaryotic histone dimers. One important difference however is that the archaeal histones lack the characteristic histone tails. The question that followed was how do these histones organize the archaeal genome? They digested chromatin thatwas extracted from Archaea with Micrococcial nuclease. This nuclease digests all DNA except the parts that are protected by histones. The digestion products were analyzed and on a gel discrete bands were observed corresponding to pieces of DNA which were a multiple of 30 base pairs in length. There were also bands corresponding to pieces of DNA longer than 120 base pairs. As each histone dimer protects approximately 30 base pairs of DNA, the results implicate that the archaeal histone-DNA complexes form a nucleosomal ramp. In contrast to the eukaryotic histones that are confined to complexes consisting of eight histones, the archaeal histones can form complexes with a large number of histones. This is a way of arranging histone-DNA complexes that has not been observed before. The hypothesis is that due to histone diversification the eukaryotic histones do not form complexes consisting of more than eight histones.
During the second part of the talk the question of how eukaryotic chromatin is propagated during DNA replication was addressed. Mattaroli is mainly interested in how the nucleosomes are disassembled and reassembled and how in this process the histone information is copied. She focusses on the role of the histone chaperone CAF-1. Histone chaperones bind to histones, thereby shielding their charges and facilitating their deposition onto the DNA. CAF-1 is known to receive an H3-H4 dimer from ASF1. However, little is known about how the chaperone actually deposit the histones on the DNA. The importance of CAF-1 is illustrated by the fact that CAF-1 is essential in multicellular organisms. It safeguards epigenome organization.
One of the outcomes of the work of Mattiroli and her colleagues was that they found the region of CAF-1 that can bind an H3-H4 dimer. This is the WD40 subunit. Once CAF-1 has a dimer bound to it, this dimer can form a tetramer. They also showed that CAF-1 can on its own assemble tetrasomes in vitro, enabling in vitro nucleosome formation. Triggered by the finding of a DNA binding winged helix domain (WHD) in CAF-1 (Zhang et al., 2016, Nucleic Acids Research) Mattiroli and her colleagues looked into the DNA binding dynamics of CAF-1. Upon binding of an H3-H4 dimer to CAF-1, the WHD is released and it can bind DNA. It was shown that WHD binds DNA in a cooperative manner. Thus it prefers to bind with two WHDs to a single piece of DNA. Once one WHD binds DNA, it triggers the binding of a second WHD and it thus triggers the binding of a second CAF-1/H3-H4 complex.
In the field there is a debate about the disassembly and reassembly of nucleosomes during replication. There are two main theories (Figure 3) about how new nucleosomes are formed. The conservative model hypothesizes that there is a separation of old and new histones after replication. The semi-conservative model on the other hand hypothesizes that old and new histones are mixed. The finding that two independent CAF-1 molecules are involved in joining two H3-H4 dimers, makes the semi-conservative model feasible. However, the majority of the evidence points to the conservative model.
Figure 3: New nucleosomes either consist of a mix of old and new histones or they consist of only old or only new histones. These two models are referred to as semiconservative and conservative respectively. Adapted from: Ramachandran, S., Henikoff, S. (2015) Replicating nucleosomes, Science Advances, Vol. 1, no. 7
There are still many questions that are left unanswered and that Mattiroli would like to address in the future. She would for example like to find how CAF-1/H3-H4 complexes are paired. There might be a role for proliferating cell nuclear antigen (PCNA) since it has multiple binding sites for CAF-1. So defining the exact role of PCNA is one of the future goals. Furthermore, Mattiroli would like to study the role of CAF-1 when the DNA has been damaged and an ultimate goal is to understand what controls epigenome inheritance.