Speaker: Fransesca Mattiroli
Department: Lugi Lab, University of Colorado Boulder
Subject: Evolution and assembly of eukaryotic Chromatin
Location: TU Delft, Bionanoscience department
Author: Mirte Golverdingen
Fransesca Mattiroli’s research is focussed on the DNA packaging units called nucleosomes. These structures organize DNA in the eukaryotic cell nucleus. Nucleosomes are formed by an octameric complex of folded histone dimers called the H3-H4 and H2A-H2B dimers. In mammals, the histones have histone tails which highly contribute to post-translational modifications and they stabilize the nucleosome. Nucleosomes need to assemble and disassemble when they bind to the genome DNA. Histone modifications and variants are dynamic and can promote or inhibit certain interactions. The nucleosome dynamics and compositions have a direct effect on transcription, translation and repair.
The first main interest of Mattiroli is the evolutionary origin of the nucleosome. The nucleosomes are very well conserved through species. Mattiroli focusses on the structural conservation of the histone dimers in Archaea. They, however, miss the tails that contribute to post-translational modification. So, how do these species organize their archaeal genome?
The archaeal histone binding to DNA is similar to eukaryotic histone binding. Archael histones, however, do not form octamers. They can form a much longer structure instead, called nucleosomal ramps. In Vivo, this structure also forms, the longest ramp they found was 90 bp long. So, they found a new way of arranging histone DNA complexes.
Histones are formed on the DNA in two steps, first, two H3-H4 dimers form a tetrasome, then two H2A-H2B dimers attach to this tetrasome forming a nucleosome. Histone chaperones shield the charges of the histones and facilitate their deposition on DNA. However, not much is known on how the chaperones actually contribute to this deposition step. The Chromatin Assembly Factor 1, CAF-1, is Mattiroli’s main interest. CAF-1 mediates in this histone deposition step and is essential in multicellular organisms. Matteroli tried to understand how CAF-1 contributes to the deposition step.
Mattiroli’s first step was to research how CAF-1 binds the H3-H4 dimer. She used mass spectrometry (HX-MS) with a hydrogen-deuterium exchange. She could, in this way, measure the change in mass and which regions have the largest changes in deuterium uptake. This region could then be the binding site of CAF-1 on H3-H4. When CAF-1 binds to the dimer, they see a stabilization of the dimer. This result indicates the following hypothesis: Only if a H3-H4 dimer is bound to CAF-1 it can form a tetrasome.
A next step for Mattiroli was to test if CAF-1 can form nucleosomes in vitro, in absence of other proteins. To test this, Mattiroli mixed CAF-1, histones and DNA, treat them with micrcococcal nuclease to digest unprotected DNA and purified and quantified the length of DNA covered by histones. The result showed that CAF-1 is able to assemble tetrasomes, and therefore enabling nucleosome formation in vitro.
So, how is the H3-H4 tetrasome on the DNA formed? Mattiroli used increased lengths of DNA, to trap any intermediates in the process. Mattiroli showed that the forming of the H3-H4 dimer activates the DNA binding of the dimer. The key intermediate that mediates the DNA binding results to be two CAF-1 units. This was the most interesting result so far, because it was never showed before that two independent CAF-1 were involved in the H3-H4 DNA binding.
The interesting and clear seminar showed again how complex the system of DNA and all the DNA-interacting molecules is. The research of Mattiroli gives a good foundation for more research to nucleosomes and their interaction with DNA. Bringing us closer to fully understand the biological system of DNA.
Figure 1. Canonical and variant nucleosomes
(A) Elements of the histone fold and structures of Xenopus leavis H2A–H2B, H3–H4 and (H3–H4)2 (PDB ID: 1KX5). (B) Structure of the canonical Xenopus leavis nucleosome (PDB ID: 1KX5). Other nucleosome structures, such as the human nucleosome, are structurally similar. (C) Structure of the CenH3CENP‐A‐containing nucleosome (PDB ID: 3AN2). (D) Zoomed view of the αN helix of CenH3CENP‐A (left) and H3 (right) involved in stabilizing the DNA ends. Histone H3 is blue, CenH3CENP‐A is cyan, H4 is green, H2A is yellow, H2B is red, and DNA is white.
Adapted From: Mattiroli, F., D’Arcy, S., & Luger, K. (2015). The right place at the right time: chaperoning core histone variants. EMBO reports, e201540840.